The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

普遍保守的Sec系统是细胞用来跨膜运输蛋白质的主要方法。直到最近，测量活性（了解生物系统如何工作的先决条件）还仅限于时间分辨力差的不连续蛋白质转运测定法，或者由干扰过程的大型非天然标记物报告。基于拆分的超亮荧光素酶（NanoLuc）的检测方法的开发改变了这一情况。在这里，我们利用这项技术来取消构成细菌中翻译后蛋白质运输的步骤。在部署的条件下，模型前蛋白底物（proSpy）的运输速度为每分钟200个氨基酸（aa），SecA能够在运输过程中解离并重新结合。在此之前，没有证据表明存在明显的限速引发事件。动力学建模表明，SecA驱动的转运活性最好通过一系列大步骤（约30 aa）来描述，每个步骤都与数百个ATP水解事件相关。我们描述的功能与不确定的运动机制（例如布朗棘轮）一致。