Syd, a SecY-interacting protein, excludes SecA from, the SecYE complex with an altered secY24 subunit
Syd, a SecY-interacting protein, excludes SecA from, the SecYE complex with an altered secY24 subunit
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DOI：
10.1074/jbc.273.30.18835
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发表时间：
1998-07-24
影响因子：
4.8
通讯作者：
Ito, K
中科院分区：
文献类型：
--
作者：
Matsuo, E;Mori, H;Ito, K
Syd是一种与SecY相互作用的大肠杆菌胞质蛋白，该蛋白的过量产生会导致许多与蛋白质易位相关的表型，包括对secY24突变细胞的强毒性。先前，这种突变被证明会破坏SecY和SecE之间的相互作用，SecY和SecE是蛋白质转座酶膜嵌入部分的两个基本亚基。目前，我们在体外研究了Syd定向抑制蛋白易位的机制，纯化的Syd蛋白可以快速抑制secY24突变细胞制备的Pro-OmpA易位到倒置膜泡（IMVs）以及伴随的SecA易位ATPase活性。在蛋白质易位过程中，携带蛋白前体的SecA与IMV上的易位酶高亲和力结合，随后atp驱动80 kda的SecA片段插入膜中。我们使用i -125标记的SecA和secY24突变体IMV进行的实验表明，Syd既消除了高亲和力的SecA结合，也消除了SecA插入。Syd甚至能够释放被一种不可水解的ATP类似物稳定的SecA插入形式。Syd明显影响了imv整合的SecY24蛋白的蛋白水解消化模式，表明Syd通过直接与SecY24蛋白相互作用发挥其抑制作用。根据这一观点，具有第二位点突变（secY249）的SecY24变体在体内和体外均抵抗Syd作用。因此，Syd作用于SecY24形式的转位酶，其中SecY24 - sece相互作用已被破坏，将SecA马达蛋白从SecYE通道复合体中排除。
Syd is an Escherichia coli cytosolic protein that interacts with SecY, Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation, Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 80-kDa SecA segment into the membrane. Our experiments using I-125-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.