Akiyama Y, Ehrmann M, Kihara A, Ito K
Department of Cell Biology, Institute for Virus Research, Kyoto University, Japan.
Mol Microbiol. 1998 May;28(4):803-12. doi: 10.1046/j.1365-2958.1998.00843.x.
The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.
大肠杆菌FtsH蛋白是一种膜结合且依赖ATP的蛋白酶。在本研究中，我们描述了FtsH中依赖ATP的构象变化以及该蛋白的多肽结合能力。在ATP存在下，FtsH的一个33 kDa片段对胰蛋白酶具有抗性。ATP和ATPγS在37℃时可防止去污剂溶解的FtsH-His6-Myc发生自我聚集，这再次表明ATP的结合会诱导FtsH发生构象变化。亲和层析显示，FtsH-His6-Myc可与变性碱性磷酸酶（PhoA）结合，但不能与天然酶结合。变性的PhoA也可防止FtsH聚集，并且这两种蛋白通过蔗糖梯度共同沉降。还证实了FtsH-His6-Myc与去污剂溶解的SecY之间的结合。尽管与FtsH结合的SecY会进一步进行依赖ATP的蛋白水解，但与FtsH结合的PhoA则不会。因此，FtsH与变性PhoA的结合与蛋白水解无关。FtsH的过量表达显著增加了MalF-PhoA杂合蛋白中PhoA部分的细胞质定位，其中在跨膜区段引入了一个带电荷的残基。因此，FtsH与变性PhoA的结合也可能在体内发生。
