An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (sigma(32)) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH(1) is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36-122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation.     

The C-terminal end of LpxC is required for degradation by the FtsH protease

Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane-bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co-purified with LpxC. However, degradation of LpxC was DnaK/J-independent in contrast to turnover of the heat shock sigma factor sigma32 (RpoH). The stability of LpxC in a bacterial one-hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C-terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half-life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C-terminus of LpxC contains a signal sequence necessary for FtsH-dependent degradation.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.     

The C terminus of sigma(32) is not essential for degradation by FtsH

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.     

Analysis of degradation of bacterial cell division protein FtsZ by the ATP-dependent zinc-metalloprotease FtsH in vitro

The identity of protease(s), which would degrade bacterial cell division protein FtsZ in vivo, remains unknown. However, we had earlier demonstrated that Escherichia coli metalloprotease FtsH degrades E. coli cell division protein FtsZ in an ATP- and Zn(2+)-dependent manner in vitro. In this study, we examined FtsH protease-mediated degradation of FtsZ in vitro in detail using seven different deletion mutants of FtsZ as the substrates, which lack different extents of specific regions at the N- or C-terminus. FtsH protease assay in vitro on these mutants revealed that FtsH could degrade all the seven deletion mutants irrespective of the deletions or the extent of deletions at the N- or C-terminus. These observations indicated that neither the N-terminus nor the C-terminus was required for the degradation of FtsZ, like already known in the case of the FtsH substrate sigma(32) protein. The recombinant clones expressing full-length FtsZ protein and FtsZ deletion mutant proteins would be useful in investigating the possibility of FtsZ as a potential in vivo substrate for FtsH in ftsH-null cells carrying ftsH suppressor function and ectopically expressed FtsH protease.     

Escherichia coli requires the protease activity of FtsH for growth

FtsH protease, the product of the essential ftsH gene, is a membrane-bound ATP-dependent metalloprotease of Escherichia coli that has been shown to be involved in the rapid turnover of key proteins, secretion of proteins into and through the membrane, and mRNA decay. The pleiotropic effects of ftsH mutants have led to the suggestion that FtsH possesses an ATP-dependent chaperone function that is independent of its protease function. When considering FtsH as a target for novel antibacterials, it is necessary to determine which of these functions is critical for the growth and survival of bacteria. To address this, we constructed the FtsH mutants E418Q, which retains significant ATPaseactivity but lacks protease activity, and K201N, which lacks both protease and ATPase activities. These mutants were introduced into an E. coli ftsH knockout strain which has wild-type FtsH supplied from a plasmid under control of the inducible araBAD promoter. Since neither mutant would complement the ftsH defect produced in the absence of arabinose, we conclude that the protease function of FtsH is required for bacterial growth.     

SpoVM, a small protein essential to development in Bacillus subtilis, interacts with the ATP-dependent protease FtsH.

The spoVM gene encodes a 26-amino-acid polypeptide that is essential for spore formation in Bacillus subtilis. A transposon insertion within the spoVM open reading frame has been shown to encode a chimeric protein which is biologically inactive and produces a phenotype identical to that of a deletion and insertion mutation. A genetic approach was used to identify possible interacting proteins, and the membrane-bound FtsH protease was identified. Mutations in ftsH suppressed the sporulation defect of certain spoVM mutants but not others. However, production of the mother cell sigma factors, sigmaE and sigmaK, was abnormal in the suppressed strains, and mutations in either spoVM or ftsH alone impaired sigma factor production and sporulation gene expression. Using FtsH purified from Escherichia coli, we demonstrated that in vitro (i) SpoVM inhibits FtsH protease activity and (ii) SpoVM is a substrate for the FtsH protease. We propose that during sporulation, SpoVM serves as a competitive inhibitor of FtsH activity. This interaction appears to be important for completion of the prespore engulfment step of sporulation, based on the phenotype of certain spoVM ftsH double mutants.