Determination of a region in SecA that interacts with presecretory proteins in Escherichia coli.
Determination of a region in SecA that interacts with presecretory proteins in Escherichia coli.
复制标题
确定 SecA 中与大肠杆菌中的前分泌蛋白相互作用的区域。
DOI：
10.1016/s0021-9258(18)38159-6
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Shoji Mizushima
中科院分区：
文献类型：
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作者：
E. Kimura;Mitsuru Akita;S. Matsuyama;Shoji Mizushima
SecA通过识别信号肽氨基末端的正电荷与分泌前蛋白相互作用（秋田，M.，Sasaki，S.，松山，S.，和Mizushima，S.（1989）J.Biol.Chem.265，8164-8169）。在6 M盐酸胍中制备多种SecA的氨基末端和羧基末端片段。然后从它们重构SecA类似物，并检查它们在1-乙基-3-（3-二甲基氨基丙基）碳二亚胺存在下与[35 S]proOmpF-Lpp（一种分泌前蛋白）交联的能力。当使用的SecA片段足够大以在结构上彼此互补时，重构的SecA类似物在与proOmpF-Lpp的交联中是有活性的。交联是信号肽依赖性的，并在其他preescretory蛋白的存在下被抑制。在ATP存在下，交联增强。然后在十二烷基硫酸钠-聚丙烯酰胺凝胶上分析与proOmpF-Lpp交联的SecA片段。交联优先发生在具有SecA的氨基末端的片段上。当羧基末端片段足够大时，也观察到弱交联。观察到交联的最小氨基末端和羧基末端片段分别为39和72 kDa。根据这些结果，推断负责与分泌前蛋白交联的区域位于SecA氨基末端的氨基酸残基267和340之间。这些结果进行了讨论有关SecA的结构和功能。
SecA interacts with presecretory proteins through recognition of the positive charge at the amino terminus of the signal peptide (Akita, M., Sasaki, S., Matsuyama, S., and Mizushima, S. (1989) J. Biol. Chem. 265, 8164-8169). A large variety of amino-terminal and carboxyl-terminal fragments of SecA were prepared in 6 M guanidine hydrochloride. SecA analogues were then reconstituted from them and examined for their ability to cross-link with [35S]proOmpF-Lpp, a presecretory protein, in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The reconstituted SecA analogues were active in the cross-linking with proOmpF-Lpp when the SecA fragments used were large enough to structurally complement each other. The cross-linking was signal peptide-dependent and suppressed in the presence of other presecretory proteins. The cross-linking was enhanced in the presence of ATP. The SecA fragments that cross-linked with proOmpF-Lpp were then analyzed on sodium dodecyl sulfate-polyacrylamide gels. The cross-linking preferentially took place on fragments possessing the amino terminus of SecA. Weak cross-linking was also observed with carboxyl-terminal fragments when they were large enough. The smallest amino-terminal and carboxyl-terminal fragments with which the cross-linking was observed were 39 and 72 kDa, respectively. From these results, the region responsible for the cross-linking with presecretory proteins was deduced to be located between amino acid residues 267 and 340 from the amino terminus of SecA. These results are discussed in relation to the structure and function of SecA.