Triplett T L, Sgrignoli A R, Gao F B, Yang Y B, Tai P C, Gierasch L M
Departments of Biochemistry & Molecular Biology and Chemistry, University of Massachusetts, Amherst, Massachusetts 01003-4510, USA.
J Biol Chem. 2001 Jun 1;276(22):19648-55. doi: 10.1074/jbc.M100098200. Epub 2001 Mar 6.
The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA. The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation. Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA. We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.
SecA对分泌前体蛋白的选择性识别是大肠杆菌蛋白质输出过程所必需的，然而，对于识别的要求以及前体与SecA的结合模式却知之甚少。主要原因是缺乏一个适合对SecA-前体复合物进行生物物理研究的可溶性系统。开发这样一个系统的复杂之处在于，只有当SecA通过与膜和SecYEG结合而被激活时，它才可能以高亲和力、有效的方式与前体相互作用。前体/SecA相互作用的一个关键方面是，它受各种SecA配体（核苷酸、脂质、SecYEG）的调节，以便促进前体的释放，很可能是以逐步的方式进行转运。最近的几份报告表明，可以使用分离的结构域来研究SecA的功能。利用这种方法，我们分离出了一个经蛋白水解产生的SecA N端片段，它折叠稳定，具有高ATP酶活性，代表了SecA的一个激活形式。我们在此报告，这个被称为SecA64的片段与信号肽的结合亲和力明显高于SecA。此外，SecA64的ATP酶活性受到信号肽的抑制，其抑制程度与这些信号肽抑制SecA转运ATP酶或体外蛋白质转运的能力相关，这表明与SecA64的相互作用在功能上具有重要意义。因此，SecA64提供了一个可溶性的、定义明确的系统，用于研究SecA对信号肽的识别模式以及信号肽释放的调节。
