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ATP依赖性蛋白酶FtsH的结构和细胞功能。FtsH形成寡聚体，并且寡聚化对其活性至关重要。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酶的六聚体环的中心孔，并且中心孔的大小在ATP水解后改变。我们已经成功地结晶的ATP酶结构域的FtsH，但X-射线衍射数据还不足以解决的晶体结构。FtsH通过降解特定的调节蛋白如sigma 32和LpxC来控制多种细胞功能。sigma 32是热休克转录因子，并被FtsH降解。FtsH对sigma 32的有效降解需要DnaK分子伴侣系统，该分子伴侣系统可以将sigma 32改变为能够接受FtsH作用的形式。我们分离到两个新的温度敏感型ftsH突变体，这些突变体的突变被定位到ftsH的C-末端区域。Mini-F质粒在温度敏感型ftsH突变体中不稳定地维持在允许细胞生长的温度下。我们还研究了λ噬菌体的裂解-溶原性决定和ftsH突变体中的大肠杆菌素耐受性。我们已经确定SpoVM作为枯草B. FtsH的底物，表明Ftsh参与孢子形成。SpoVM作用于膜并对细胞生长产生抑制作用。

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.