SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo.
SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo.
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DOI：
10.1128/jb.00622-16
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发表时间：
2017-01-15
影响因子：
3.2
通讯作者：
Bukau B
中科院分区：
文献类型：
--
作者：
Huber D;Jamshad M;Hanmer R;Schibich D;Döring K;Marcomini I;Kramer G;Bukau B
SecA是细菌中Sec机制的重要组成部分，负责跨细胞质膜转运蛋白质。我们实验室最近的工作表明SecA与核糖体结合。在这里，我们使用了两种不同的方法来证明SecA也与新生多肽在体内相互作用，这些多肽是Sec底物。首先，我们在体内将SecA光交联到核糖体上，并鉴定出与SecA共纯化的mRNA。共纯化mRNA的微阵列分析表明含有SEC靶向序列的蛋白质的强烈富集。其次，我们使用了2维（2-D）凝胶方法来分析放射性标记的新生多肽，与SecA，包括麦芽糖结合蛋白，一个良好的特征SecA底物共纯化。SecA与新生链的相互作用在缺乏SecB或触发因子的细胞中没有受到强烈影响，这两者也与新生Sec底物相互作用。事实上，SecB与新生链相互作用的能力在SecA与核糖体之间的相互作用有缺陷的菌株中被破坏。SecA与含有体外捕获的新生链的纯化核糖体的相互作用的分析表明，SecA可以开始与各种新生链相互作用，当它们达到1/110个氨基酸的长度时，这比与SecB相互作用所需的长度短得多。我们的研究结果表明，SecA coconferentially承认新生的SEC基板，这种识别可能需要这些蛋白质的有效交付的膜嵌入式SEC机器。重要性SecA是一种ATP酶，为细菌中的Sec机制跨细胞质膜转运蛋白质提供能量。大多数这些蛋白质的易位是从蛋白质合成中解偶联的，通常被描述为“翻译后”。在这里，我们表明，SecA与新生的SEC底物相互作用。这种相互作用不依赖于SecB或触发因子，它们也与新生的Sec底物相互作用。此外，SecB与新生多肽的相互作用依赖于SecA与核糖体的相互作用，这表明新生链与SecA的相互作用先于与SecB的相互作用。我们的研究结果表明，SecA可以协同识别底物蛋白，以便有效地靶向它们进行解偶联蛋白易位。
SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery. IMPORTANCE SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as “posttranslational.” Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.