Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Proc Natl Acad Sci U S A. 2013 Sep 17;110(38):E3650-9. doi: 10.1073/pnas.1306738110. Epub 2013 Sep 3.
The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.
双精氨酸转运（Tat）机制将折叠蛋白穿过细菌的细胞质膜和叶绿体的类囊体膜进行转运。据推断，Tat 易位位点是由底物诱导的 TatA 蛋白的结合按需组装而成的。我们通过在低水平 TatA 同源物 TatE 存在的情况下，在活的大肠杆菌细胞中以天然水平表达的 YFP 标记 TatA 进行成像，来测试这个模型。在这些条件下，TatA-YFP 融合支持完整的生理 Tat 转运活性。与 TatA 结合模型一致，在细胞内增加有转运能力的底物蛋白的数量会导致存在的大 TatA 复合物数量增加。这些复合物的形成需要功能正常的 TatBC 底物受体和跨膜质子动力势（PMF）。去除 PMF 会导致 TatA 复合物解离，除非在 Tat 转运活性受损的菌株中。基于这些观察结果，我们提出 TatA 组装达到了一个临界点，在这个临界点上，只有通过底物转运才能逆转寡聚化。与 TatA-YFP 不同，TatB-YFP 和 TatC-YFP 融合蛋白的寡聚状态不受底物或 PMF 的影响，尽管 TatB-YFP 寡聚化确实需要 TatC。
