ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase. To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP). HslVU degraded both proteins upon incubation with ATP but not with its nonhydrolyzable analog, ATPgammaS, indicating that the degradation of SulA requires ATP hydrolysis. The pulse-chase experiment using an antibody raised against MBP-SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon). These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.     

The ATP-Dependent HslVU/ClpQY Protease Participates in Turnover of Cell Division Inhibitor SulA in Escherichia coli

Escherichia coli mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [DeltahslVU and Delta(clpPX-lon)], cannot grow at low (30 degrees C) or very high (45 degrees C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30 degrees C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either ftsZ (encoding a key cell division protein) or sulA (encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the ftsZ mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the sulA mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Delta(clpPX-lon) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.     

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage.     

Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.     

Lon protease promotes survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic glucose starvation

In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Aerobic induction of the Clp family of ATP-dependent proteases could explain these results, but direct quantitation of Clp protein established that its level was not affected by Lon deficiency and overexpression of Clp did not rescue the cells under anaerobic conditions. We conclude that the Lon protease supports survival during anaerobic carbon starvation by a mechanism which does not depend on Clp. Shen Luo and Megan McNeill contributed equally to this research.     

Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (-/-) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I-containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.     

Determination of the cleavage sites in SulA, a cell division inhibitor, by the ATP-dependent HslVU protease from Escherichia coli

HslVU is an ATP-dependent protease from Escherichia coli and known to degrade SulA, a cell division inhibitor, both in vivo and in vitro, like the ATP-dependent protease Lon. In this study, the cleavage specificity of HslVU toward SulA was investigated. The enzyme was shown to produce 58 peptides with various sizes (3-31 residues), not following the 'molecular ruler' model. Cleavage occurred at 39 peptide bonds preferentially after Leu in an ATP-dependent manner and in a processive fashion. Interestingly, the central and C-terminal regions of SulA, which are known to be important for the function of SulA, such as inhibition of cell division and molecular interaction with certain other proteins, were shown to be preferentially cleaved by HslVU, as well as by Lon, despite the fact that the peptide bond specificities of the two enzymes were distinct from each other.     

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.     

DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate

The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase gamma and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.     

Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.     

Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli

The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.     

ATP依赖的人Lon蛋白酶的表达、纯化及其活性鉴定

ATP依赖的人Lon蛋白酶是一种同质寡聚、环状的蛋白酶,主要位于细胞线粒体基质中。许多研究表明,Lon蛋白酶对于维护细胞的内环境稳定起着重要作用,并参与线粒体蛋白质量控制和代谢调控。将pPROEX1 His6-Lon重组质粒在Escherichia coli Rosetta 2菌株中诱导表达用Ni2＋柱亲和层析法纯化,获得纯度较高的目的蛋白。经纯化后,Lon蛋白酶的比酶活达到0.17 U/mg。通过多肽底物Rhodamine 110、bis-（CBZ-L-alanyl-L-alanine amide）[（Z-AA）2 Rh110]的降解检测显示,Lon蛋白酶具有肽酶活性,并被ATP所刺激。Casein和线粒体转录因子A降解实验表明,纯化的Lon蛋白酶具有蛋白水解活性,而且蛋白水解活性依赖于ATP。     

Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease.

Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro

To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells.     

The ClpPX protease is required for radioresistance and regulates cell division after gamma-irradiation in Deinococcus radiodurans

Protein degradation in bacteria is involved in diverse cellular responses to environmental stimuli and in removing potentially toxic damaged proteins or protein aggregates. ATP-dependent proteases play a key role in these processes. Here, we have individually inactivated all the ATP-dependent proteases belonging to the Clp or Lon families in Deinococcus radiodurans. The mutants were tested for survival after gamma-irradiation and for sensitivity to the tRNA analogue puromycin in order to assess the impact of each disruption on radioresistance, as well as on proteolysis of misfolded proteins. We found that inactivation of the ClpPX protease significantly decreased cell survival at elevated gamma-irradiation doses, while inactivation of Lon1 and Lon2 proteases reduced resistance to puromycin, suggesting that they play a role in eliminating damaged proteins. Mutants devoid of ClpPX protease displayed altered kinetics of DNA double-strand break repair and resumed cell division after an exceedingly long lag phase following completion of DNA repair. During this stasis period, most of the DeltaclpPX irradiated cells showed decondensed nucleoids and abnormal septa and some cells were devoid of DNA. We propose that the ClpPX protease is involved in the control of proper chromosome segregation and cell division in cells recovering from DNA damage.     

Proteolysis and modulation of the activity of the cell division inhibitor SulA in Escherichia coli lon mutants.

Intracellular accumulation of the inducible cell division inhibitor SulA is modulated by proteases that ensure its degradation, namely, the Lon protease and another ATP-dependent protease(s). Lon- cells are UV sensitive because SulA is stable. We asked whether these ATP-dependent proteases are more active when lon cells are grown at high temperature or in synthetic medium since these conditions decrease the UV sensitivity of lon cells. We found that these growth conditions have no direct effect on Lon-independent degradation of SulA. They may, instead, decrease the SulA-FtsZ interaction.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ⁻ cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon. Received: 8 October 1996 / Accepted: 27 November 1996     

The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli.

SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In this study, we investigated the cleavage specificity of Lon protease toward SulA protein. The enzyme was shown to cleave approximately 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1, and P1' positions, respectively, and one or two Gln residues in positions P2-P5. They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in alpha helix and beta sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.