Membrane protein degradation by E. coli FtsH

大肠杆菌FtsH是一种膜结合和atp依赖的蛋白酶。FtsH的n端区域介导膜结合以及该酶的同质寡聚相互作用。之前我们研究了FtsH衍生物的体内功能，其中n端膜区域要么被删除（FtsH（ΔTM）），要么被亮氨酸拉链（Zip-FtsH（ΔTM））取代，要么被乳糖渗透酶跨膜段（LacY-FtsH）取代(1)。研究表明，最低蛋白水解活性需要同源寡聚化，而膜蛋白降解需要跨膜序列。我们在体外纯化并鉴定了这些蛋白。LacY-FtsH可以降解可溶性蛋白和膜蛋白，而Zip-FtsH（ΔTM）只降解可溶性蛋白。这些蛋白也表现出显著的atp酶活性。然而，FtsH（ΔTM）在atp酶和蛋白酶活性方面保持失活，尽管它保留了atp结合和变性蛋白结合能力。这些结果表明，亚基结合对于FtsH的ATP水解活性是重要的，并且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。我们发现这种酶也可以在胞质尾部的c端启动蛋白水解。这种降解方式也是渐进的，可以被紧密折叠的质周区域终止。这些结果表明，同一酶可以表现出N到C或C到N的加工性。FtsH的序列比对表明，谷氨酸残基在大肠杆菌FtsH的Glu 479对应位置保守。这个位置的突变损害了体内FtsH的蛋白水解功能。突变体酶的体外蛋白水解活性较低，但在高浓度锌离子的刺激下活性显著提高。突变没有引起FtsH的总体构象变化。突变蛋白纯化后锌含量降低。结果表明，Glu 479是锌配位残基。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