Plant Biology Department, University of California, Davis, CA 95616.
Plant Biology Department, University of California, Davis, CA 95616
Proc Natl Acad Sci U S A. 2021 Mar 23;118(12). doi: 10.1073/pnas.2018122118.
The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between "closed" and "opened" states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.
在折叠底物转运过程中，Tat 复合物的机制和孔结构仍然是一个谜，部分原因是转运后迅速解离。相比之下，蛋白质 SecY 孔是一种持久的结构，在转运未折叠底物链时只需要在“关闭”和“打开”状态之间经历构象转变。虽然蛋白质孔模型很好地描述了 SecY 孔，但环形孔模型更好地解释了在 Tat 转运中穿过疏水性双层转运折叠底物时必须克服的高能量障碍。原则上，膜电导行为可用于区分环形孔和蛋白质孔，如许多抗菌肽以及线粒体 Bax 和 Bid 的检测所示。在这里，我们测量了电致变色位移（ECS）衰减作为分离类囊体中电导的替代物，既在蛋白质转运期间，也在组成型组装的转运子中进行。我们发现，组成型组装 Tat 复合物的膜和正在进行 Tat 转运的膜显示出与静止膜相似的电导特性。进行 Sec 转运的膜和与底物结合的 SecY 孔的膜导致电场衰减明显更快。在具有活性 SecY 的膜中 ECS 信号的响应性使人想起蛋白质孔中施加电压与电导之间的陡峭关系，而在相同电场下，Tat 转运和组成型 Tat 复合物的非加速电场衰减与环形孔的行为一致。
