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Membrane protein degradation by E. coli FtsH

大肠杆菌 FtsH 降解膜蛋白

基本信息

批准号：
11680697
负责人：
AKIYAMA Yoshinori
金额：
$ 2.3万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680697/
关键词：
E. coli FtsH ATPase Protein degradation membrane protein AAA family Protein degradation プロテアーゼ膜ポテンシャル膜貫通配列

项目摘要

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

大肠杆菌 FtsH 是一种膜结合且 ATP 依赖性蛋白酶。 FtsH 的 N 末端区域介导膜缔合以及该酶的同源寡聚体相互作用。之前我们研究了 FtsH 衍生物的体内功能，其中 N 端膜区域被删除 (FtsH(ΔTM))，被亮氨酸拉链 (Zip-FtsH(ΔTM)) 取代，或被乳糖通透酶跨膜片段 (LacY-FtsH) 取代 (1)。这表明同源寡聚化是最小蛋白水解活性所必需的，而跨膜序列是膜蛋白降解所必需的。我们在体外纯化并表征了这些蛋白质。 LacY-FtsH 降解可溶性蛋白和膜蛋白，但 Zip-FtsH(ΔTM) 仅降解可溶性蛋白。这些蛋白质还表现出显着的 ATP 酶活性。然而，FtsH(ΔTM) 在 ATP 酶和蛋白酶活性方面仍然不活跃，尽管它保留了 ATP 结合以及变性的蛋白质结合能力。这些结果表明，亚基关联对于 FtsH 的 ATP 水解活性很重要，并且即使在去污剂溶解的条件下，跨膜序列也必须存在才能降解膜蛋白。我们证明这种酶还可以在 C 端胞质尾部启动蛋白水解。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，相同的酶可以表现出 N 到 C 或 C 到 N 的持续合成能力，具体取决于。 FtsH 的序列比对表明，与大肠杆菌 FtsH 的 Glu 479 对应的位置处的谷氨酸残基是保守的。该位置的突变损害了 FtsH 体内的蛋白水解功能。突变酶的体外蛋白水解活性较低，但高浓度锌离子显着刺激。这些突变并未引起 FtsH 的总体构象变化。纯化后突变蛋白表现出锌含量降低。从这些结果中，我们得出结论：Glu 479 是锌配位残基。较少的