| Research Abstract |
We have studied an essential, membrane-bound ATP-dependent protease, FtsH in E.coli. FtsH controls several cellular functions by degrading specific regulatory proteins such as sigma32 and LpxC.Sigma32 is the heat shock transcription factor. Efficient degradation of sigma32 by FtsH requires the DnaK chaperone system, which may alter sigma32 to a form competent to the FtsH action. Loss of the ftsH function results in stabilization of LpxC, the committed enzyme involved in lipid A synthesis, leading overproduction of lipopolysaccharide. Under such conditions, abnormally layered membrane structures are formed in the periplasmic space. Certain mutations, which upregulate phospholipid synthesis, suppresses both lethality of ftsH mutations and formation of abnormal membrane structures. Mini-F plasmids are unstably maintained in temperature-sensitive ftsH mutants at permissive temperatures for cell growth. We have also studied colicin tolerance in ftsH mutants. FtsH forms oligomers, and oligomerizaton is essential for its activity. N-terminal transmembrane and periplasmic domains, in particular the second transmembrane domain, are important for oligomerization. Site-directed mutagenesis and homology modeling revealed that the ATP-binding pocket is formed at the interface of neighboring subunits, and that invariant residues in the AAA ATPase-specific sequence, SRH, play important roles in ATP hydrolysis. These results have led us to propose an intermolecular catalysis model for the AAA ATPase. We have also analyzed functional motifs of an AAA^+ protein, RuvB, and investigated similarities and differences between AAA and AAA^+ ATPases.
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