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.     

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.     

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).     

DnaK-sigma 32 interaction is temperature-dependent. Implication for the mechanism of heat shock response

The heat shock response in bacteria is a complex phenomenon in which sigma 32 plays the central role. The DnaK/J chaperone system binds and promotes degradation of sigma 32 at lower temperatures. At heat shock temperatures, the DnaK/J-mediated degradation of sigma 32 is largely abolished by a mechanism, which is not yet fully understood. In this article we have shown that interaction of DnaK with sigma 32 is highly temperature-dependent. This interaction is completely abolished at 42 degrees C. To investigate the origin of such strong temperature dependence, we have monitored the structural changes that occur in the sigma 32 protein upon upshift of temperature and attempted to elucidate its functional roles. Upon a shift of temperature from 30 to 42 degrees C, the CD spectrum of sigma 32 becomes significantly more positive without significant change in either tryptophan fluorescence spectra or quenchability to external quenchers. 1,8-Anilinonaphthalene sulfonic acid binding at 42 degrees C is not significantly affected. The equilibrium guanidine hydrochloride denaturation of sigma 32 is biphasic. The first phase shifts to even lower guanidine hydrochloride concentrations at 42 degrees C, whereas the major phase remains largely unchanged. The sigma 32-core interaction remains unchanged as a function of temperature. This suggests that increased temperature destabilizes a structural element. We discuss the possible location of this temperature-sensitive structural element.     

In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes.

In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product. Previous work has shown that the HtpR protein serves as a sigma factor (sigma 32) for RNA polymerase that specifically recognizes heat shock promoters (A.D. Grossman, J.W. Erickson, and C.A. Gross Cell 38:383-390, 1984). In the present study we showed that sigma 32 synthesized in vitro could stimulate the expression of heat shock genes. The in vitro-synthesized sigma 32 was found to be associated with RNA polymerase. In vivo-synthesized sigma 32 was also associated with RNA polymerase, and this polymerase (E sigma 32) could be isolated free of the standard polymerase (E sigma 70). E sigma 32 was more active than E sigma 70 with heat shock genes; however, non-heat-shock genes were not transcribed by E sigma 32. The in vitro expression of the htpR gene required E sigma 70 but did not require E sigma 32.