Translocation reversal by the thylakoid twin-arginine translocase

真核细胞包含许多不同的隔间，这些隔间被紧密密封的生物膜所包围。前往这些隔室的蛋白质需要通过膜运输，这是由一系列蛋白质运输系统进行的。它们通常通过展开蛋白质并将其穿过细胞膜上的一个狭窄小孔来发挥作用。然而，叶绿体类囊体膜上的一个主要的蛋白质转位酶，被称为双精氨酸转位(TAT)系统，使用一种独特的机制将大的、完全折叠的蛋白质穿过这一膜输送到管腔空间。以前使用分离膜的研究表明，该系统可以高效地运输蛋白质，而且始终是单向的。运输后，运输蛋白上的信号肽在管腔中被移除，以产生成熟蛋白。我们现在发现了这个系统的另一个非常不寻常的特征；在完整的活细胞中对相同底物的分析表明，底物通常只被部分跨膜运输，之后它们被返回基质并降解。尤其是在信号肽与外来蛋白质相连的结构中，几乎所有的蛋白质都经历了这个“易位逆转”的过程。这可能反映了质量控制或“校对”活动的操作，由此TAT设备仅识别并完全传送正确类型的折叠结构。这是系统活动中一个意想不到的关键部分，我们建议调查其根本原因和后果。第二个主要目的是利用我们工作的另一个发现：当信号肽的切割被阻止时，底物蛋白在易位过程中被困在易位酶中(逆转位极大地减慢)。这使我们有机会研究底物在被转移的过程中，并首次识别和表征实际的易位孔。反过来，这将导致重要的机械论见解。

Eukaryotic cells contain numerous different compartments that are bounded by tightly sealed biological membranes. Proteins destined for these compartments need to be transported across the membranes and this is carried out by a range of protein transport systems. These usually function by unfolding the protein and 'threading' it through a narrow pore in the membrane. However, a major protein translocase in the chloroplast thylakoid membrane, termed the twin-arginine translocation (Tat) system, uses a unique mechanism to transport large, fully-folded proteins across this membrane into the lumenal space. Previous studies using isolated membranes have shown that the system transports proteins with high efficiency, and invariably in one direction. After transport, the signal peptides on the transported proteins are removed in the lumen to create the mature protein. We have now discovered another highly unusual trait of this system; analysis of the same substrates in intact living cells shows that substrates are often transported only partially across the membrane, after which they are returned to the stroma and degraded. This is especially the case with a construct in which the signal peptide is linked to a 'foreign' protein, where almost all of the protein undergoes this process of 'translocation reversal'. This may reflect the operation of a quality control, or 'proofreading' activity, whereby only the correct type of folded structure is recognized and fully transported by the Tat apparatus. This is an unexpected and critical part of the system's activity and we propose to investigate the underlying causes and consequences. The second main aim is to capitalise on another finding of our work: when cleavage of the signal peptide is prevented, the substrate protein becomes trapped in the translocase during translocation (retrotranslocation is slowed down enormously). This gives us an opportunity to study the substrate in the process of being translocated, and to identify and characterise the actual translocation pore for the first time. This, in turn, will lead to important mechanistic insights.