Huber Damon, Jamshad Mohammed, Hanmer Ruby, Schibich Daniela, Döring Kristina, Marcomini Isabella, Kramer Günter, Bukau Bernd
Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
J Bacteriol. 2016 Dec 28;199(2). doi: 10.1128/JB.00622-16. Print 2017 Jan 15.
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.
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.
SecA是细菌Sec转运机制的一个必需组分，负责将蛋白质转运穿过细胞质膜。我们实验室最近的研究表明SecA与核糖体结合。在此，我们采用两种不同方法来证明SecA在体内也与新生多肽相互作用，并且这些多肽是Sec底物。首先，我们在体内将SecA与核糖体进行光交联，并鉴定与SecA共纯化的mRNA。对共纯化mRNA的微阵列分析表明，含有Sec靶向序列的蛋白质有很强的富集。其次，我们采用二维（2-D）凝胶方法分析与SecA共纯化的放射性标记新生多肽，包括麦芽糖结合蛋白，一种特征明确的SecA底物。在缺乏SecB或触发因子的细胞中，SecA与新生链的相互作用并未受到强烈影响，SecB和触发因子也都与新生Sec底物相互作用。实际上，在SecA与核糖体之间的相互作用存在缺陷的菌株中，SecB与新生链相互作用的能力被破坏。体外分析SecA与含有停滞新生链的纯化核糖体之间的相互作用表明，当各种新生链达到约110个氨基酸的长度时，SecA就可以开始与它们相互作用，这比与SecB相互作用所需的长度要短得多。我们的结果表明，SecA在共翻译过程中识别新生Sec底物，并且这种识别可能是将这些蛋白质有效递送至膜嵌入的Sec转运机制所必需的。
SecA是一种ATP酶，为细菌中的Sec转运机制将蛋白质转运穿过细胞质膜提供能量。这些蛋白质中的大多数转运与蛋白质合成解偶联，并且经常被描述为“翻译后”。在此，我们表明SecA与新生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。我们的结果表明，SecA可以在共翻译过程中识别底物蛋白，以便有效地将它们靶向解偶联的蛋白质转运。
