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是膜结合的和ATP依赖性蛋白酶。 FTSH的N末端区域介导了该酶的膜关联以及同性恋的相互作用。以前，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Zipper（Zip-ftsh（ΔTM））代替，或者由乳糖固定酶跨膜段（lacy-ftsh）代替。据表明，最小蛋白水解活性需要同型寡聚化，而膜蛋白降解需要跨膜序列。我们在体外纯化并表征了这些蛋白质。 Lacy-ftsh降解了固体和膜蛋白，但Zip-ftsh（ΔTM）仅降解固体蛋白。这些蛋白质还暴露了重要的ATPase活性。然而，尽管它保留了ATP结合以及变性的蛋白质结合能力，但FTSH（ΔTM）在ATPase和蛋白酶活性中仍然不活跃。这些结果INDI…更多类别对FTSH的ATP水解活性很重要，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。我们表明，该酶也可以在C末端胞质尾部启动蛋白水解。这种降解模式也是过程的，可以通过紧密折叠的外围浆细胞域流产。这些结果表明，相同的酶可以表现出N至C或C到N的n个过程。 FTSH的序列比对表明，谷氨酸的固定在与大肠杆菌FTSH的GLU 479相对应的位置保守。该位置的突变损害了FTSH体内FTSH的蛋白水解功能。突变酶的体外蛋白水解活性较低，但高浓度的锌离子显着刺激。突变并未引起FTSH的严重构象变化。突变蛋白在纯化后暴露于减少锌含量。从这些结果中，我们包含GLU 479是锌协调的住所。较少的