Protein quality control: triage by chaperones and proteases.
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ATP-Dependent Proteases↗
Biomedical subjects
S Gottesman
Publications and source records attributed to S Gottesman.
Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY.
ClpQ (HslV) is a homolog of the beta-subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HslU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit Mr 19,000) and ClpY (subunit Mr 49,000) were purified separately as oligomeric proteins with molecular weights of approximately 220,000 and approximately 350,000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP-dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six-fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry-matched ClpYQ system.
ATP-Dependent Proteases↗
Role of the heat shock protein DnaJ in the lon-dependent degradation of naturally unstable proteins.
We have investigated the role of DnaJ in protein degradation by examining the degradation of intrinsically unstable proteins by Lon protease in vivo. In Escherichia coli, Lon protease is responsible for the rate-limiting step in degradation of highly unstable proteins such as SulA, RcsA, and lambdaN protein, as well as for about 50% of the rapid degradation of abnormal proteins such as canavanine-containing proteins. We found that Lon-dependent degradation of both SulA and lambdaN protein was unaffected in cells lacking functional DnaJ, whereas Lon-dependent turnover of canavanine-containing proteins was slower in dnaJ mutant cells. DnaJ also affected the slow SulA degradation seen in the absence of Lon. The rate of degradation of RcsA was reduced in dnaJ mutants, but both Lon-dependent and Lon-independent degradation was affected; abnormal, canavanine-containing proteins were similarly affected. Both the RcsA that accumulated in dnaJ mutant cells and the SulA that accumulated in lon dnaJ mutant cells was aggregated. The abnormal proteins that partitioned to the insoluble pellet became solubilized over time in dnaJ+ cells but not in dnaJ- cells. Therefore, the co-chaperone DnaJ is not essential for Lon-dependent degradation and may act in protein turnover only as an accessory factor helping to maintain substrates in a soluble form. Such an accessory factor is apparently necessary for abnormal proteins and for RcsA. The relative rates of degradation and aggregation of specific protein targets may determine the importance of the chaperone systems in turnover of a given protein.
ATP-Dependent Proteases↗
ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions.
CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (Km >/=200 microM), and the peptide bond turnover number was approximately 10 min-1. CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity. CcdA lost alpha-helical structure at elevated temperatures (Tm approximately 50 degrees C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.
ATP-Dependent Proteases↗
The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli.
dsrA encodes a small, untranslated RNA. When over-expressed, DsrA antagonizes the H-NS-mediated silencing of numerous promoters. Cells devoid of DsrA grow normally and show little change in the expression of a number of H-NS-silenced genes. Expression of a transcriptional fusion of lacZ to dsrB, the gene next to dsrA, is significantly lower in cells carrying mutations in dsrA. All expression of beta-galactosidase from the dsrB::lacZ fusion is also dependent on the stationary phase sigma factor, RpoS. DsrA RNA was found to regulate dsrB::lacZ indirectly, by modulating RpoS synthesis. Levels of RpoS protein are substantially lower in a dsrA mutant, both in stationary and exponential phase cells. Mutations in dsrA decrease the expression of an RpoS::LacZ translational fusion, but not a transcriptional fusion, suggesting that DsrA is acting after transcription initiation. While RpoS expression is very low in exponential phase at temperatures of 30 degrees C and above, at 20 degrees C there is substantial synthesis of RpoS during exponential growth, all dependent on DsrA RNA. dsrA expression is also increased at low temperatures. These results suggest a new role for RpoS during exponential growth at low temperatures, mediated by DsrA.
Bacterial Proteins↗
Osmotic shock induction of capsule synthesis in Escherichia coli K-12.
The genes (cps) involved in the synthesis of the colanic acid capsular polysaccharide in Escherichia coli K-12 are transcriptionally regulated by numerous proteins. Two of these, RcsB and RcsC, share homology with two-component regulatory elements that respond to environmental stimuli. Osmotic shock by sucrose or NaCl transiently increased transcription of a cpsB::lacZ fusion. RcsC and RcsB were essential for osmotic induction of colanic acid synthesis. In contrast to observations in some other osmotically regulated systems, addition of glycine betaine enhanced the osmotic induction of cps::lacZ by both sucrose and NaCl but had no effect alone.
Bacterial Capsules↗
Proteases and their targets in Escherichia coli.
Proteolysis in Escherichia coli serves to rid the cell of abnormal and misfolded proteins and to limit the time and amounts of availability of critical regulatory proteins. Most intracellular proteolysis is initiated by energy-dependent proteases, including Lon, ClpXP, and HflB; HflB is the only essential E. coli protease. The ATPase domains of these proteases mediate substrate recognition. Recognition elements in target are not well defined, but are probably not specific amino acid sequences. Naturally unstable protein substrates include the regulatory sigma factors for heat shock and stationary phase gene expression, sigma 32 and RpoS. Other cellular proteins serve as environmental sensors that modulate the availability of the unstable proteins to the proteases, resulting in rapid changes in sigma factor levels and therefore in gene transcription. Many of the specific proteases found in E. coli are well-conserved in both prokaryotes and eukaryotes, and serve critical functions in developmental systems.
Endopeptidases↗
A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli.
The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA. The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis. We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter. A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing. DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E. coli. Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation.
ATP-Dependent Proteases↗
Selective, energy-dependent proteolysis in Escherichia coli.
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ATP-Dependent Proteases↗
A molecular chaperone, ClpA, functions like DnaK and DnaJ.
The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK). Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.
Adenosine Triphosphatases↗
Alp suppression of Lon: dependence on the slpA gene.
We have previously found that plasmids carrying the Escherichia coli alp gene (now to be called alpA) suppress two phenotypes of a delta lon protease mutant, overproduction of capsular polysaccharide and sensitivity to UV light. Suppression of these lon phenotypes is most likely explained by the increased degradation of the Lon substrates responsible for these phenotypes. We have called this suppressing protease activity Alp protease. The Alp protease activity is detected in cells after introduction of plasmids carrying the alpA gene, which encodes an open reading frame of 70 amino acids. Insertions which abolish Alp activity interrupt this open reading frame. We have used Tn10 and lambda placMu mutagenesis to identify a chromosomal locus, slpA, that is required for alpA+ suppression of delta lon. This locus maps at 57 min, close to the chromosomal location of alpA. The expression of beta-galactosidase from a lac transcriptional fusion to slpA is increased six- to eightfold when the alpA+ gene is present on a multicopy plasmid. Therefore, AlpA acts as a transcriptional regulator of the slpA gene(s); activation of slpA transcription is necessary to suppress the phenotypes of a delta lon mutation. In an accompanying paper (J. E. Kirby, J. E. Trempy, and S. Gottesman, J. Bacteriol. 176:2068-2081, 1994), we show that neither AlpA nor SlpA is a component of the protease itself but that they are part of a regulatory cascade which leads to expression of the Alp protease.
ATP-Dependent Proteases↗
Excision of a P4-like cryptic prophage leads to Alp protease expression in Escherichia coli.
The Escherichia coli K-12 alpA gene product, when overproduced from a multicopy plasmid, leads to suppression of the capsule overproduction and UV sensitivity phenotypes of cells mutant for the Lon ATP-dependent protease. This suppression has previously been shown to correlate with increased in vivo activity of a previously unknown energy-dependent proteolytic activity capable of degrading Lon substrates, the Alp protease. We show in an accompanying paper that alpA, which has homology to a short open reading frame in bacteriophage P4, acts as a positive transcriptional regulator of slpA, a gene linked to alpA and necessary for suppression of lon mutants (J. E. Trempy, J. E. Kirby, and S. Gottesman, J. Bacteriol. 176:2061-2067). The sequence of slpA suggests that it encodes an integrase gene closely related to P4 int and that both alpA and slpA are part of a cryptic P4-like prophage. AlpA expression increases SlpA synthesis. Increased SlpA leads, in turn, to the excision and loss of the cryptic prophage. Excision is dependent on integration host factor as well as on SlpA. Prophage excision is necessary but not sufficient for full expression of the Alp protease. A second function (named AHA) allows full protease expression; this function can be provided by the kanamycin resistance element from Tn903 when the element is present on a multicopy plasmid. Excision and loss of the cryptic prophage apparently allow expression of the Alp protease by inactivating a small stable RNA (10Sa RNA) encoded by the ssrA gene. The precursor of this RNA has its 3' end within the cryptic prophage; the mature 3' end lies within the prophage attL site. Inactivation of ssrA by insertional mutagenesis is sufficient to allow expression of the suppressing Alp protease, even in the presence of the cryptic prophage. Therefore, 10Sa RNA acts as a negative regulator of protease synthesis or activity, and prophage excision must inactivate this inhibitory function of the RNA.
Amino Acid Sequence↗
An imbalance of HU synthesis induces mucoidy in Escherichia coli.
Mutations in a number of loci, including the lon gene, dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid. The lon gene, which encodes an ATP-dependent protease, is localized at ten minutes on the E. coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU. Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E. coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid. As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically. Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis. Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting. The UV sensitivity of the hup transformed lon+ bacteria is identical to the lon+ parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal. Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.
ATP-Dependent Proteases↗
A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.
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 cell 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 cells. A truncated LON gene, in which translation was initiated at Met118 of the coding sequence, was expressed in Escherichia coli and produced a protease that degraded alpha-casein in vitro in an ATP-dependent manner and had other properties similar to E. coli Lon protease.
ATP-Dependent Proteases↗
ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities.
The ATP-dependent Clp protease of Escherichia coli consists of two subunits, the ClpP subunit, which has the proteolytic active site, and ClpA, which possesses ATPase activity and activates the proteolytic activity of ClpP in vitro. Recently, Zylicz and co-workers (Wojtkowiak, D., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 22609-22617) identified another E. coli protein that activated ATP-dependent degradation of lambda O protein in the presence of ClpP. The amino-terminal sequence of this protein corresponds to the translated amino-terminal sequence of a gene that we have named clpX. clpX encodes a protein with M(r) 46,300, similar to that observed for the protein purified by Wojtkowiak et al. clpX is an operon with clpP; both genes are cotranscribed in a single heat-inducible 2200-base mRNA, with clpP the promoter proximal gene. The sequence of ClpX includes a single consensus ATP-binding site motif and has limited homology to regions of ClpA and other members of the ClpA/B/C family. A third group of proteins, ClpY, closely related to ClpX, has been identified by sequence homology. Mutations in either clpX or clpP abolish degradation of the highly unstable lambda O protein in vivo. clpX mutants are not defective in degradation of previously identified ClpA/ClpP substrates such as a ClpA-beta-galactosidase fusion protein. It appears that selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits.
ATP-Dependent Proteases↗
Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.
Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.
Carbohydrate Sequence↗
Regulation by proteolysis: energy-dependent proteases and their targets.
A number of critical regulatory proteins in both prokaryotic and eukaryotic cells are subject to rapid, energy-dependent proteolysis. Rapid degradation combined with control over biosynthesis provides a mechanism by which the availability of a protein can be limited both temporally and spatially. Highly unstable regulatory proteins are involved in numerous biological functions, particularly at the commitment steps in developmental pathways and in emergency responses. The proteases involved in energy-dependent proteolysis are large proteins with the ability to use ATP to scan for appropriate targets and degrade complete proteins in a processive manner. These cytoplasmic proteases are also able to degrade many abnormal proteins in the cell.
ATP-Dependent Proteases↗
Uses of transposons with emphasis on Tn10.
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Bacteriophage lambda↗