TRANSLOCATION CAN DRIVE THE UNFOLDING OF A PREPROTEIN DOMAIN
TRANSLOCATION CAN DRIVE THE UNFOLDING OF A PREPROTEIN DOMAIN
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DOI：
10.1002/j.1460-2075.1993.tb05650.x
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发表时间：
1993-01-01
期刊：
影响因子：
11.4
通讯作者：
WICKNER, W
中科院分区：
文献类型：
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作者：
ARKOWITZ, RA;JOLY, JC;WICKNER, W
前体蛋白被认为在穿过大肠杆菌质膜易位之前具有二级和三级结构。我们现在发现前蛋白在易位过程中的解折叠可以由易位事件本身驱动。在某些阶段，易位和解折叠可以在没有外源能量输入的情况下发生。为了检查这种解折叠反应，我们制备了proOmpA-Dhfr，这是一种充分研究的细胞溶质酶二氢叶酸还原酶（Dhfr）与proOmpA（外膜蛋白A的前体形式）C-末端连接的融合蛋白。在其体外易位的中间阶段，N-末端proOmpA结构域已经穿过膜，而折叠的Dhfr部分，由其配体NADPH和甲氨蝶呤稳定，没有。当从该中间体中除去配体时，通过两步过程发生易位。首先，融合蛋白的20-30个氨基酸残基伴随Dhfr结构域的解折叠而易位。该反应既不需要ATP、DELTAmu（H）+也不需要转位酶的SecA亚基。引人注目的是，这种易位加速了Dhfr结构域的净展开。在第二步中，SecA和ATP水解驱动易位的快速完成。因此，来自易位的能量可以驱动大量蛋白质结构域的解折叠。
Precursor proteins are believed to have secondary and tertiary structure prior to translocation across the Escherichia coli plasma membrane. We now rind that preprotein unfolding during translocation can be driven by the translocation event itself. At certain stages, translocation and unfolding can occur without exogenous energy input. To examine this unfolding reaction, we have prepared proOmpA-Dhfr, a fusion protein of the well studied cytosolic enzyme dihydrofolate reductase (Dhfr) connected to the C-terminus of proOmpA, the precursor form of outer membrane protein A. At an intermediate stage of its in vitro translocation, the N-terminal proOmpA domain has crossed the membrane while the folded Dhfr portion, stabilized by its ligands NADPH and methotrexate, has not. When the ligands are removed from this intermediate, translocation occurs by a two-step process. First, 20-30 amino acid residues of the fusion protein translocate concomitant with unfolding of the Dhfr domain. This reaction requires neither ATP, DELTAmu(H)+ nor the SecA subunit of translocase. Strikingly, this translocation accelerates the net unfolding of the Dhfr domain. In a second step, SecA and ATP hydrolysis drive the rapid completion of translocation. Thus energy derived from translocation can drive the unfolding of a substantial protein domain.