ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus
ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus
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DOI：
10.1128/jb.00802-16
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发表时间：
2017-04-01
影响因子：
3.2
通讯作者：
Pohlschroder, Mechthild
中科院分区：
文献类型：
--
作者：
Halim, Mohd Farid Abdul;Stoltzfus, Jonathan D.;Pohlschroder, Mechthild
大多数原核细胞分泌的蛋白质通过一般分泌（Sec）或双精氨酸易位（Tat）途径转运到细胞表面。大多数分泌的蛋白质固定在细胞表面，而其余的则释放到细胞外环境中。锚定的表面蛋白在细胞过程中发挥着各种重要作用，从促进细胞之间的相互作用到维持细胞稳定性。被广泛研究的火山卤黄的s层糖蛋白（SLG），以前被认为是通过c端嵌入到膜上，最近被证明是脂化的，并且其c端片段在依赖于新发现的酶——古选酶A （ArtA）的过程中被移除。虽然SLG是一个Sec底物，但本文的计算机分析显示，在预测的8个额外的ArtA底物中，有两个底物也含有预测的Tat信号肽，包括Hvo_0405，它具有高度保守的三重结构，位于蛋白质中心而不是其C端，这与迄今为止确定的其他预测的ArtA底物不同。我们证明，即使给定其非典型位置，这种三方结构可能是由编码ArtA底物和细胞质蛋白的基因融合造成的，以依赖于ArtA的方式进行加工。利用Hvo_0405突变体缺乏预测的Tat信号肽的保守“双”精氨酸，我们发现Hvo_0405确实是Tat底物，而ArtA底物包括Sec和Tat底物。最后，我们利用质谱法确定了Hvo_0405的at依赖性定位和信号肽酶I （SPase I）裂解位点。促进蛋白质锚定在古细菌细胞表面的具体机制仍然知之甚少。在这里，我们已经证明，通过最近发现的一种新的依赖于arta的锚定机制，结合在模型古细菌H. volcanii细胞表面的蛋白质在结构上比以前已知的更加多样化。具体来说，我们的研究结果表明，Tat和Sec底物都含有预测的ArtA底物的保守三方结构，可以以依赖于ArtA的方式进行加工，并且三方结构不需要位于C端附近就可以进行加工。这些数据提高了我们对古细菌细胞生物学的理解，对于古细菌和细菌蛋白质的亚细胞定位预测是无价的。
Most prokaryote-secreted proteins are transported to the cell surface using either the general secretion (Sec) or twin-arginine translocation (Tat) pathway. A majority of secreted proteins are anchored to the cell surface, while the remainder are released into the extracellular environment. The anchored surface proteins play a variety of important roles in cellular processes, ranging from facilitating interactions between cells to maintaining cell stability. The extensively studied S-layer glycoprotein (SLG) of Haloferax volcanii, previously thought to be anchored via C-terminal intercalation into the membrane, was recently shown to be lipidated and to have its C-terminal segment removed in processes dependent upon archaeosortase A (ArtA), a recently discovered enzyme. While SLG is a Sec substrate, in silico analyses presented here reveal that, of eight additional ArtA substrates predicted, two substrates also contain predicted Tat signal peptides, including Hvo_0405, which has a highly conserved tripartite structure that lies closer to the center of the protein than to its C terminus, unlike other predicted ArtA substrates identified to date. We demonstrate that, even given its atypical location, this tripartite structure, which likely resulted from the fusion of genes encoding an ArtA substrate and a cytoplasmic protein, is processed in an ArtA-dependent manner. Using an Hvo_0405 mutant lacking the conserved "twin" arginines of the predicted Tat signal peptide, we show that Hvo_0405 is indeed a Tat substrate and that ArtA substrates include both Sec and Tat substrates. Finally, we confirmed the Tat-dependent localization and signal peptidase I (SPase I) cleavage site of Hvo_0405 using mass spectrometry.IMPORTANCE The specific mechanisms that facilitate protein anchoring to the archaeal cell surface remain poorly understood. Here, we have shown that the proteins bound to the cell surface of the model archaeon H. volcanii, through a recently discovered novel ArtA-dependent anchoring mechanism, are more structurally diverse than was previously known. Specifically, our results demonstrate that both Tat and Sec substrates, which contain the conserved tripartite structure of predicted ArtA substrates, can be processed in an ArtA-dependent manner and that the tripartite structure need not lie near the C terminus for this processing to occur. These data improve our understanding of archaeal cell biology and are invaluable for in silico subcellular localization predictions of archaeal and bacterial proteins.