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).     

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.     

Marked instability of the sigma(32) heat shock transcription factor at high temperature. Implications for heat shock regulation.

The heat shock response in Escherichia coli depends on a transient increase in the intracellular level of sigma(32) that results from both increased synthesis and transient stabilization of normally unstable sigma(32). Although the membrane-bound ATP-dependent protease FtsH (HflB) plays an important role in degradation of sigma(32), our previous results suggested that several cytosolic ATP-dependent proteases including HslVU (ClpQY) are also involved in sigma(32) degradation (Kanemori, M., Nishihara, K., Yanagi, H., and Yura, T. (1997) J. Bacteriol. 179, 7219-7225). We now report on the ATP-dependent proteolysis of sigma(32) by purified HslVU protease and its unusual dependence on high temperature: sigma(32) was rapidly degraded at 44 degrees C, but with much slower rates ( approximately 15-fold) at 35 degrees C. FtsH-dependent degradation of sigma(32) also gave similar results. In agreement with these results in vitro, the turnover of sigma(32) in normally growing cells at high temperature (42 degrees C) was much faster than at low temperature (30 degrees C). Taken together with other evidence, these results suggest that the sigma(32) level during normal growth is primarily determined by the stability (susceptibility to proteases) and synthesis rate of sigma(32) set by ambient temperature, whereas fine adjustment such as transient stabilization of sigma(32) observed upon heat shock is brought about through monitoring changes in the cellular state of protein folding.     

Escherichia coli HflK and HflC can individually inhibit the HflB (FtsH)-mediated proteolysis of λCII in vitro

λCII is the key protein that influences the lysis/lysogeny decision of λ by activating several phage promoters. The effect of CII is modulated by a number of phage and host proteins including Escherichia coli HflK and HflC. These membrane proteins copurify as a tightly bound complex ‘HflKC’ that inhibits the HflB (FtsH)-mediated proteolysis of CII both in vitro and in vivo. Individual purification of HflK and HflC has not been possible so far, since each requires the presence of the other for proper folding. We report the first purification of HflK and HflC separately as active and functional proteins and show that each can interact with HflB on its own and each inhibits the proteolysis of CII. They also inhibit the proteolysis of E. coli σ³² by HflB. We show that at low concentrations each protein is dimeric, based on which we propose a scheme for the mutual interactions of HflB, HflK and HflC in a supramolecular HflBKC protease complex.     

A colicin-tolerant Escherichia coli mutant that confers hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (HflB)

An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype.     

Characterization of multi-cellulases of Sporotrichum cellulophilum by using monoclonal antibodies

Cellulases of Sporotrichum cellulophilum, CII1, CII2, CII3, CII5, CIII1, CIII2, and CIV1 were purified to homogeneity by column chromatographies and preparative electrophoresis. The specific activity of CII3 was the highest (100 units/mg), while the activities of CII5, CIII1, and CII2 were low (12–17 units/mg). The molecular weight of CII2 was 19,000, while those of the others ranged from 46,000–63,000. All the cellulases were glycoproteins except for CII2. Three monoclonal antibodies (Mab CII11, Mab CII12, and MabCII13) were prepared against CII1, and Mab CIV11 against CIV1. From the immunological reactivities, it was found that CII1, CIII2, and CIV1 had a common antigenic site recognized by Mab CII11, Mab CII12, and Mab CII13, while CII12 and CVI1 had a different antigenic site that was recognized by Mab CIV11, MabCII11, MabCII12, MabCII13, and MabCIV11 did not react with CII2, CII3, CII5, and CIII1. The classification of these cellulases was discussed.