Functions and Fine Structure of the AAA Protease FtsH

我们研究了大肠杆菌中一种必需的、膜结合的atp依赖蛋白酶FtsH的结构和细胞功能。FtsH形成低聚物，低聚对其活性至关重要。位点定向突变和同源性建模表明，ATP结合袋是在邻近亚基的界面上形成的，并且AAA ATP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六聚体AAA atp酶的分子间催化模型。该模型还表明，未折叠的底物穿过ATP酶六聚体环的中心孔，并且中心孔的大小在ATP水解时发生改变。我们已经成功地结晶了FtsH的atp酶结构域，但是x射线衍射数据还不足以解析晶体结构。FtsH通过降解特定的调节蛋白如sigma32和LpxC来控制几种细胞功能。Sigma32是热休克转录因子，可被FtsH降解。FtsH对sigma32的有效降解需要DnaK伴侣系统，该系统可能将sigma32改变为能够发挥FtsH作用的形式。我们新分离了两个温度敏感的ftsH突变体，并将这些突变体的突变定位到ftsH的c端区域。在温度敏感的ftsH突变体中，在允许细胞生长的温度下，Mini-F质粒是不稳定的。我们还研究了ftsH突变体中λ噬菌体的溶原性决定和粘菌素耐受性。我们已经确定SpoVM是枯草芽孢杆菌FtsH的底物，表明FtsH参与孢子形成。SpoVM作用于细胞膜，对细胞生长有抑制作用。

We have studied structure and cellular functions of an essential, membrane-bound ATP-dependent protease, FtsH in E.coli. FtsH forms oligomers, and oligomerization is essential for its activity. 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 hexameric AAA ATPase. The model also suggests that unfolded substrates are threaded through the central hole of the hexameric ring of the ATPase, and that the size of the central hole is altered upon ATP hydrolysis. We have succeeded to crystallize the ATPase domain of FtsH, however X-ray diffraction data have not been sufficient to resolve the crystal structure. FtsH controls several cellular functions by degrading specific regulatory proteins such as sigma32 and LpxC.Sigma32 is the heat shock transcription factor and is degraded by FtsH.Efficient degradation of sigma32 by FtsH requires the DnaK chaperone system, which may alter sigma32 to a form competent to the FtsH action. We isolated two temperature-sensitive ftsH mutants newly, and mutations of these mutants were mapped to the C-terminal region of ftsH.Mini-F plasmids are unstably maintained in temperature-sensitive ftsH mutants at permissive temperatures for cell growth. We have also studied lysis-lysogeny decision of lambda phage and colicin tolerance in ftsH mutants. We have identified SpoVM as a substrate of B.subtilis FtsH, suggesting Ftsh's involvement in sporulation. SpoVM acts for the membrane and exerts an inhibitory effect on cell growth.