Miller A, Wang L, Kendall D A
Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269, USA.
J Biol Chem. 1998 May 8;273(19):11409-12. doi: 10.1074/jbc.273.19.11409.
Although it is known that virtually all exported proteins require a signal peptide, it is not clearly understood how the signal peptide interfaces with the translocation machinery to achieve transport. In this study we document a direct interaction between the signal peptide and SecA, a primary component of the translocase in Escherichia coli, and show that the signal peptide itself can stimulate SecA-lipid ATPase activity. Using synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and two model sequences, we find that the extent of stimulation of SecA ATPase activity by the different peptides parallels the hierarchy of results found for in vivo function (Izard, J. W., Doughty, M. B., and Kendall, D. A. (1995) Biochemistry 34, 9904-9912). The peptide-induced activity requires a lipid to protein molar ratio of at least 300:1 and liposomes enriched in negatively charged phospholipids. Furthermore, specific binding of the signal peptide to SecA was demonstrated using chemical cross-linking and competition with unlabeled peptides.
虽然已知几乎所有输出蛋白都需要信号肽，但信号肽如何与转运机制相互作用以实现转运尚不清楚。在本研究中，我们记录了信号肽与SecA（大肠杆菌转位酶的主要成分）之间的直接相互作用，并表明信号肽本身可以刺激SecA-脂质ATP酶活性。使用对应于野生型碱性磷酸酶信号序列和两个模型序列的合成信号肽，我们发现不同肽对SecA ATP酶活性的刺激程度与体内功能的结果层次平行（伊扎德，J.W.，道蒂，M.B.，和肯德尔，D.A.（1995年）《生物化学》34，9904-9912）。肽诱导的活性需要脂质与蛋白质的摩尔比至少为300:1以及富含带负电荷磷脂的脂质体。此外，使用化学交联和与未标记肽的竞争证明了信号肽与SecA的特异性结合。
