A CYTOPLASMIC DOMAIN IS IMPORTANT FOR THE FORMATION OF A SECY-SECE - TRANSLOCATOR COMPLEX
A CYTOPLASMIC DOMAIN IS IMPORTANT FOR THE FORMATION OF A SECY-SECE - TRANSLOCATOR COMPLEX
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DOI：
10.1073/pnas.91.10.4539
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发表时间：
1994-05-10
影响因子：
11.1
通讯作者：
ITO, K
中科院分区：
文献类型：
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作者：
BABA, T;TAURA, T;ITO, K
确定多组分蛋白质组装的相互作用位点的方法已被应用于膜结合的SecY-SecE复合物，其介导蛋白质跨大肠杆菌细胞质膜的输出。显性负性secY等位基因secY（-d）1使SecY失活，但保留其与SecE相互作用的能力。因此，突变蛋白以无活性复合物螯合SecE。破坏SecE结合位点的第二位点突变将抑制输出干扰。我们引入了基因内抑制secY（-d）1的插入/缺失突变。在通过表达已连接到C末端的LacZ α序列消除敲除突变后，我们在胞质结构域4中获得了惊人的突变聚类。基于该结果，检查了该结构域中的secY 24（Ts）置换突变对与SecE相互作用的影响。它确实抑制了secY（-d）1。虽然与过量SecY相关的不稳定性可以通过过量产生SecE来减轻，但是SecY 24突变蛋白不被SecE稳定。基础水平的SecY 24蛋白在42 ℃下也不稳定。SecE与SecY（+）共免疫沉淀，但不与SecY 24蛋白共免疫沉淀。这些结果表明secY 24突变减弱了SecY与SecE的相互作用。两者合计，我们建议，胞质结构域4是重要的SecY和SecE之间的关联。
An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane. A dominant negative secY allele, secY(-d)1, inactivates SecY but preserves its ability to interact with SecE. Thus, the mutant protein sequesters SecE in an inactive complex. Second site mutations that disrupt the SecE binding site will suppress the export interference. We introduced insertion/deletion mutations that intragenically suppressed secY(-d)1. After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4. On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE. It indeed suppressed secY(-d)1. Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE. The basal-level SecY24 protein was also destabilized at 42 degrees C. SecE was coimmunoprecipitated with SecY(+) but not with the SecY24 protein. These results indicate that the secY24 mutation weakens SecY's interaction with SecE. Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE.