The TatA subunit of Escherichia coli twin-arginine translocase has an N-in topology
The TatA subunit of Escherichia coli twin-arginine translocase has an N-in topology
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DOI：
10.1021/bi7005288
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发表时间：
2007-06-26
期刊：
影响因子：
2.9
通讯作者：
Turner, Raymond J.
中科院分区：
文献类型：
--
作者：
Chan, Catherine S.;Zlomislic, Marian R.;Turner, Raymond J.
双精氨酸移位酶（达特）系统被许多细菌用于跨细胞质膜移位折叠蛋白。TatA亚基是预测的成孔亚基，并已显示形成同源寡聚复合物。通过巯基反应试剂4-乙酰氨基-4 '-马来酰亚胺基二苯乙烯-2，2'-二磺酸和N-α-（3-马来酰亚胺基丙酰基）生物胞素对TatA中位点特异性半胱氨酸突变体的可及性实验，我们表明TatA的N-末端位于细胞质中，而不是先前假设的周质中。我们也证实了以前的观察，C-末端有一个双重拓扑结构。通过处理与膜解偶联剂羰基氰-间氯苯腙，我们表明，C-末端的拓扑状态是依赖于膜电位。这些结果表明两种架构的TatA在膜：一个与一个单一的跨膜螺旋和其他两个跨膜螺旋。这两种拓扑结构的分子模型被用来开发和卡通同源寡聚复合物作为一个通道，直径类似于50 A，并建议双跨膜螺旋拓扑结构可能是易位通道的构建模块。此外，Gly 2Cys和Thr 22 Cys突变体的体内交联实验表明，Gly 2在跨膜螺旋-1的开始处与相邻TatA的Gly 2非常接近，因为Cys 2在加入铜菲咯啉后立即交联。另一方面，Cys 22，在跨膜螺旋的另一端，需要至少10分钟的交联，这表明可能的运动或重新定位是必需的，使该残基接近相邻的TatA亚基。
The twin-arginine translocase (Tat) system is used by many bacteria to translocate folded proteins across the cytoplasmic membrane. The TatA subunit is the predicted pore-forming subunit and has been shown to form a homo-oligomeric complex. Through accessibility experiments using the thiol-reactive reagents 4-acetamido-4 '-maleimidylstilbene-2,2 '-disulfonic acid and N-alpha-(3-maleimidylproprionyl)biocytin toward site-specific cysteine mutants in TatA, we show that the N-terminus of TatA is located in the cytoplasm rather than the previously assumed periplasm. We also confirm previous observations that the C-terminus has a dual topology. By treatment with the membrane uncoupler carbonyl cyanide-m-chlorophenyl hydrazone, we show that the topological state of the C-terminus is dependent on the membrane potential. These results suggest two architectures of TatA in the membrane: one with a single transmembrane helix and the other with two transmembrane helices. Molecular models of both topologies were used to develop and cartoon a homo-oligomeric complex as a channel with a diameter of similar to 50 A and suggest that the double transmembrane helix topology might be the building block for the translocation channel. Additionally, in vivo cross-linking experiments of Gly2Cys and Thr22Cys mutants showed that Gly2, at the beginning of transmembrane helix-1, is in close proximity with Gly2 of a neighboring TatA, as Cys2 cross-linked immediately upon the addition of copper phenanthroline. On the other hand, Cys22, at the other end of the transmembrane helix, took at least 10 min to cross-link, suggesting that a possible movement or reorientation is required to bring this residue into proximity with a neighboring TatA subunit.