Sakoh Machiko, Ito Koreaki, Akiyama Yoshinori
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
J Biol Chem. 2005 Sep 30;280(39):33305-10. doi: 10.1074/jbc.M506180200. Epub 2005 Aug 2.
Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.
大肠杆菌HtpX是一种推测的膜结合锌金属蛋白酶，有人认为它与FtsH（一种膜结合且依赖ATP的蛋白酶）共同参与膜蛋白的蛋白水解质量控制。在此，我们对HtpX进行了生化特性分析，并证实了其对膜蛋白和可溶性蛋白的蛋白水解活性。HtpX在细胞破碎或膜溶解时会发生自我降解。因此，我们在变性条件下纯化了HtpX，然后在锌螯合剂存在的情况下将其重折叠。当补充Zn2+时，纯化后的酶表现出自我切割活性。在有锌存在的情况下，它还能降解酪蛋白并切割一种可溶性膜蛋白SecY。我们通过过量表达HtpX和SecY来验证其在体内切割SecY的能力。这些结果表明，HtpX是大肠杆菌膜定位蛋白水解系统中锌依赖性内切蛋白酶成员。
