Background

The Sec translocon moves proteins across lipid bilayers in all domains of life. In bacteria, a heterotrimeric transmembrane channel (SecYEG), located in the plasma membrane, associates with an ATPase, SecA, which drives substrate protein translocation. SecA utilizes chemical energy from ATP to drive the reaction forward and help to destabilize the tertiary structure of the substrate protein that otherwise interferes with translocation progress. Substrate proteins pass through the channel in an unfolded conformation.

The exact mechanisms underlying translocon function have remained unclear despite decades of study. Mechanistic studies of Sec-dependent protein translocation have traditionally been carried out using protease protection experiments (Cunningham et al., 1989). Substrate proteins are imported into vesicles or proteoliposomes containing reconstituted translocon complexes. The addition of a non-specific protease to the reaction results in proteolysis of untranslocated polypeptides, whereas proteins translocated into the interior of the proteoliposomes are protected from digestion. The protected undigested proteins are subsequently quantitated after separation by gel electrophoresis. These measurements have been instrumental in elucidating the function of the Sec translocon [see for instance (Brundage et al., 1990; Schiebel et al., 1991; van der Wolk et al., 1997; Mao et al., 2013; Bauer et al., 2014; Mao et al., 2020)]. However, the time resolution of this assay is limited because samples need to be taken and processed individually for each timepoint; thus, detailed kinetic information about the translocation process cannot be obtained.

Recently, an assay for continuously monitoring translocation has been described (Pereira et al., 2019). This assay is based on complementation of a highly active luciferase, NanoLuc, which can be asymmetrically split into a large (18 kDa) and small (1.3 kDa) fragment, termed 11S and p86, respectively (Dixon et al., 2016). Neither fragment alone exhibits significant luciferase activity. When 11S is encapsulated into SecYEG/SecA proteoliposomes, complete translocation of a substrate protein C-terminally tagged with p86 restores luciferase activity. The high affinity of the 11S/p86 complex (K_D = 700 pM (Dixon et al., 2016)) and the high catalytic activity after association of the fragments permits sensitive detection of substrate proteins accumulating inside the proteoliposomes after complete translocation.

We have combined the luminescence-based real-time translocation assay (Pereira et al., 2019) with a method of generating reversibly stalled translocation intermediates (Erlandson et al., 2008) to kinetically resolve Sec-dependent protein translocation (Gupta et al., 2020). Together with a detailed kinetic model of the underlying process (Gupta et al., 2020), this approach has allowed us to dissect individual components of the process. Here, we describe the protocols for these measurements, which should be useful for obtaining mechanistic insight into the function of the Sec translocon.

The methodology described here is ideally suited for mechanistic studies of Sec translocon activity. The continuous measurement of substrate import provides time resolution that is superior to that of the traditional protease protection assays. Together with the model that we developed for analysis, the approach enables detailed kinetic dissection of the process, providing mechanistic insight into the SecA function.