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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

E. coli FtsH是一种膜结合的ATP依赖性蛋白酶。FtsH的N-末端区域介导该酶的膜缔合以及同源寡聚体相互作用。此前，我们研究了FtsH衍生物的体内功能，其中N末端膜区域被缺失（FtsH（ΔTM））、被亮氨酸拉链（Zip-FtsH（ΔTM））替换或被乳糖通透酶跨膜片段（LacY-FtsH）替换（1）。结果表明，同源寡聚化是必需的最低蛋白水解活性，而跨膜序列是必需的膜蛋白降解。我们在体外纯化并鉴定了这些蛋白。LacY-FtsH降解可溶性蛋白和膜蛋白，而Zip-FtsH（ΔTM）仅降解可溶性蛋白。这些蛋白也表现出显着的ATP酶活性。而FtsH（ΔTM）虽然保留了ATP结合和变性蛋白结合的能力，但在ATP酶和蛋白酶活性上均无活性。这些结果表明， ...更多信息表明亚基缔合对于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，跨膜序列也必须存在以降解膜蛋白。我们发现，这种酶也可以启动蛋白质水解的C-末端胞质尾部。这种降解方式也是进行性的，可以通过紧密折叠的周质结构域中止。这些结果表明，同一种酶可以表现出N到C或C到N的持续合成能力。FtsH的序列比对表明，谷氨酸残基在对应于E. coli FtsH.该位置的突变损害了体内FtsH的蛋白水解功能。在体外的突变酶的蛋白水解活性低，但显着刺激高浓度的锌离子。突变没有引起FtsH的总体构象变化。纯化后，突变体蛋白质表现出降低的锌含量。从这些结果，我们得出结论，Glu 479是一个锌配位残基。少