Membrane protein degradation by E. coli FtsH

E.ColiFtsH是一种膜结合型、依赖于ATP的蛋白酶。FtsH的N-末端区域介导该酶的膜结合和同源低聚相互作用。在此之前，我们研究了FtsH衍生物的体内功能，其中N-端膜区被删除(FtsH(ΔTM))，被亮氨酸拉链(ZIP-FtsH(ΔTM))取代，或者被乳糖渗透酶跨膜片段(Lacy-FtsH)取代(1)。结果表明，蛋白降解活性最低需要同源齐聚，而膜蛋白降解则需要跨膜序列。我们在体外对这些蛋白进行了纯化和鉴定。Lacy-FtsH对可溶性蛋白和膜蛋白均有降解作用，而Zip-FtsH(ΔTM)仅对可溶性蛋白有降解作用。这些蛋白质还显示出显著的ATPase活性。然而，FtsH(ΔTM)虽然保留了与三磷酸腺苷结合和变性蛋白结合的能力，但在三磷酸腺苷酶和蛋白水解酶活性上都保持不活跃。这些结果指示…更多的证据表明，亚基结合对FtsH的ATP水解性很重要，即使在洗涤剂溶解的条件下，也必须存在跨膜序列才能降解膜蛋白。我们发现，这种酶还可以在C末端胞浆尾部启动蛋白分解。这种降解模式也是进行性的，可以通过紧密折叠的周质结构域来中止。这些结果表明，同一种酶可以表现出N到C或C到N的加工性。FtsH的序列比对表明，谷氨酸残基在与E.ColiFtsH的Glu 479对应的位置是保守的。该位置的突变损害了体内FtsH的蛋白分解功能。突变体酶的体外蛋白水解酶活性较低，但受到高浓度锌离子的显著刺激。这些突变没有引起FtsH的严重构象变化。突变蛋白纯化后锌含量降低。根据这些结果，我们认为Glu 479是一个锌配位残基。较少

E. coli FtsH is a membrane-bound and ATP-dependent protease. The N-terminal region of FtsH mediates membrane association as well as homo-oligomeric interaction of this enzyme. Previously we studied in vivo functionality of FtsH derivatives, in which the N-terminal membrane region was either deleted (FtsH(ΔTM)), replaced by a leucine-zipper (Zip-FtsH(ΔTM)), or replaced by a lactose permease transmembrane segment (LacY-FtsH)(1). It was indicated that homo-oligomerization is required for the minimum proteolytic activity, whereas a transmembrane sequence is required for membrane protein degradation. We purified and characterized these proteins in vitro. LacY-FtsH degraded both soluble and membrane proteins, but Zip-FtsH(ΔTM) only degraded soluble proteins. These proteins also exhibited significant ATPase activities. However, FtsH(ΔTM) remained inactive both in ATPase and protease activities, although it retained ATP-binding as well as denatured protein-binding abilities. These results indi … More cates that subunit association is important for the ATP hydrolytic activity of FtsH, and that the transmembrane sequence must exist to degrade a membrane protein even under detergent-solubilized conditions. We showed that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail. This mode of degradation is also processive, which can be aborted by a tightly folded periplasmic domain. These results suggest that a same enzyme can exhibits either N to C or C to N processivity depending. Sequence alignment of FtsH indicates that glutamic acid residues are conserved at the positions corresponding to Glu 479 of E. coli FtsH. Mutations at this position compromised the proteolytic functions of FtsH in vivo. In vitro proteolytic activities of the mutant enzymes were low but significantly stimulated by high concentration of zinc ion. The mutations did not cause gross conformational changes in FtsH. The mutant proteins exhibited reduced zinc contents upon purification. From these results, we conclude that Glu 479 is a zinc-coordinating residue. Less