AAA Protease FtsH Controlling a Variety of Cellular Functions

我们已经研究了一种必需的，膜结合ATP依赖性蛋白酶，FtsH在大肠杆菌。FtsH通过降解特异性调节蛋白如sigma 32和LpxC来控制多种细胞功能。FtsH对sigma 32的有效降解需要DnaK分子伴侣系统，该系统可将sigma 32改变为能够胜任FtsH作用的形式。ftsH功能的丧失导致参与脂质A合成的关键酶LpxC的稳定，导致脂多糖的过度产生。在这样的条件下，在周质空间中形成异常分层的膜结构。某些上调磷脂合成的突变抑制ftsH突变的致死性和异常膜结构的形成。Mini-F质粒在允许细胞生长的温度下不稳定地维持在温度敏感的ftsH突变体中。我们还研究了ftsH突变体中的大肠杆菌素耐受性。FtsH形成寡聚体，并且寡聚化对其活性是必需的。N-末端跨膜和周质结构域，特别是第二跨膜结构域，对于寡聚化是重要的。定点突变和同源性建模表明，ATP结合口袋形成于相邻亚基的界面处，并且AAA ATP酶特异性序列中的不变残基SRH在ATP水解中起重要作用。这些结果使我们提出了AAA ATP酶的分子间催化模型。我们还分析了AAA^+蛋白RuvB的功能基序，并研究了AAA和AAA^+ ATP酶之间的异同。

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.