Huang Qi, Palmer Tracy
Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, United Kingdom
mBio. 2017 Aug 1;8(4):e00909-17. doi: 10.1128/mBio.00909-17.
The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway. Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.
一般分泌途径（Sec）和双精氨酸转运酶（Tat）并行运作，将蛋白质输出原核生物的细胞质膜和植物叶绿体的类囊体膜。底物通过具有三方结构的N端信号肽靶向各自的机制；然而，Tat信号肽含有一对保守且几乎不变的精氨酸，这对于有效靶向Tat机制至关重要。Tat信号肽与由TatB和TatC组件组成的膜结合受体复合物相互作用，TatC含有双精氨酸识别位点。在这里，我们在Tat底物SufI的信号肽中分离出了抑制子，这些抑制子在TatC双精氨酸结合位点存在失活替代的情况下恢复了Tat转运。这些抑制子增加了信号肽的疏水性，共纯化实验表明它们恢复了与变体TatBC复合物的结合。疏水性抑制子也可以在体内发挥作用，抑制信号肽双精氨酸基序处的替代，这些替代通常会阻止靶向Tat途径。含有四个亮氨酸替代的SufI信号肽的高度疏水性变体保留了与Tat系统相互作用的能力。含有双赖氨酸残基的两个Sec底物DsbA和OmpA的疏水性信号肽被证明可介导Tat途径的输出并与TatBC共纯化。这些发现表明Sec和Tat信号肽之间存在前所未有的重叠，并且信号肽双精氨酸基序和TatC双精氨酸识别位点都不是Tat途径运作的必需机制特征。蛋白质输出是所有原核生物中的一个基本过程。Sec和Tat输出途径并行运作，Sec机制运输无结构的前体，Tat途径运输折叠的蛋白质。蛋白质通过含有几乎不变的双精氨酸基序的N端信号肽靶向Tat途径。在这里，我们有一个惊人发现，即双精氨酸对于Tat机制识别底物并非必不可少，并且通过增加信号肽的疏水性可以绕过这一要求。我们进一步表明，真正的Sec底物的信号肽也可以介导Tat途径的运输。我们的发现表明，Tat靶向机制的关键特征已经进化，以防止底物错误靶向Sec途径，而不是Tat途径功能的关键要求。
