Teru Ogura

AAA+ superfamily ATPases: common structure–diverse function

The AAA+ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring‐shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring‐shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA–protein complexes. Thus, the AAA+ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA+ ATPases and other ring‐shaped ATPases.

The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino‐terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha‐helical coiled‐coil domains and one globular domain, and a carboxyl‐terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild‐type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.

Dissecting the Role of a Conserved Motif (the Second Region of Homology) in the AAA Family of ATPases SITE-DIRECTED MUTAGENESIS OF THE ATP-DEPENDENT PROTEASE FtsH

Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain ofN-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.

Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the σ32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes σ32, and the FtsH protease, which is largely responsible for σ32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose‐controlled promoters. Small increases in levels of DnaK and the DnaJ co‐chaperone (< 1.5‐fold of wild type) resulted in decreased level and activity of σ32 at intermediate temperature and faster shut‐off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat‐denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut‐off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the σ32 levels, but the accumulated σ32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress‐sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.

E.coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA binding and ATP/GTP binding activities.

mukB mutants of Escherichia coli are defective in the correct partitioning of replicated chromosomes. This results in the appearance of normal‐sized anucleate (chromosome‐less) cells during cell proliferation. Based on the nucleotide sequence of the mukB gene, the MukB protein of 177 kDa was predicted to be a filamentous protein with globular domains at the ends, and also having DNA binding and nucleotide binding abilities. Here we present evidence that the purified MukB protein possesses these characteristics. MukB forms a homodimer with a rod‐and‐hinge structure having a pair of large, C‐terminal globular domains at one end and a pair of small, N‐terminal globular domains at the opposite end; it tends to bend at a middle hinge site of the rod section. Chromatography in a DNA‐cellulose column and the gel retardation assay revealed that MukB possesses DNA binding activity. Photoaffinity cross‐linking experiments showed that MukB binds to ATP and GTP in the presence of Zn2+. Throughout the purification steps, acyl carrier protein was co‐purified with MukB.

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R‐3‐hydroxyacyl‐ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42°C. This was accompanied by a dramatic increase in the amount of UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse‐chase experiments and in vitro assays with purified components showed that FtsH, the AAA‐type membrane‐bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl‐ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R‐3‐hydroxyacyl‐ACP pool, is regulated by FtsH.

Recent advances in p97/VCP/Cdc48 cellular functions ☆

p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases.

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage λ CII transcriptional activator plays a key role in the lysis‐lysogeny decision by phage λ. Here we demonstrate that the E. coli ATP‐dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat‐shock transcription factor σ32. Proteolysis of σ32 requires, in vivo, the presence of the DnaK‐DnaJ‐GrpE chaperone machine. Neither DnaK‐DnaJ‐GrpE nor GroEL‐GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP‐dependent proteolysis of CII in vitro. The degradation of CII is at least 10‐fold faster than that of σ32. Electron microscopy revealed that purified FtsH forms ring‐shaped structures with a diameter of 6–7 nm.

Conserved Pore Residues in the AAA Protease FtsH Are Important for Proteolysis and its Coupling to ATP Hydrolysis

Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe228 and Gly230) that are predicted to lie in the central pore region of the hexamer. The functions of Phe228 and Gly230 were probed by site-directed mutagenesis. The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228. None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade σ32, a physiological substrate. The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not. Mutation of these two pore residues also affected the ATPase activity of FtsH. The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity. The F228E mutant was actually more active ATPase. The substrates, σ32 and casein, stimulated the ATPase activity of wild type FtsH. The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe228 mutants, but not the G230A mutant, remained σ32-stimulatable. These results suggest that Phe228 and Gly230 in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis.

The crystal structure of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli at 1.5 Å resolution

Eubacteria and eukaryotic cellular organelles have membrane-bound ATP-dependent proteases, which degrade misassembled membrane protein complexes and play a vital role in membrane quality control. The bacterial protease FtsH also degrades an interesting subset of cytoplasmic regulatory proteins, including sigma(32), LpxC, and lambda CII. The crystal structure of the ATPase module of FtsH has been solved, revealing an alpha/beta nucleotide binding domain connected to a four-helix bundle, similar to the AAA modules of proteins involved in DNA replication and membrane fusion. A sulfate anion in the ATP binding pocket mimics the beta-phosphate group of an adenine nucleotide. A hexamer form of FtsH has been modeled, providing insights into possible modes of nucleotide binding and intersubunit catalysis.