In bacteria, proteins are secreted across the cytoplasmic membrane by a protein complex termed translocase. The ability to study the activity of the translocase in vitro using purified proteins has been instrumental for our understanding of the mechanisms underlying this process. Here, we describe the protocols for the purification and reconstitution of the SecYEG complex in an active state into liposomes. In addition, fluorescence based in vitro assays are described that allow monitoring translocation activity discontinuously and in real time.
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Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation.
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Article Synopsis
- - The Sec61 translocon is critical for transporting newly formed proteins into the endoplasmic reticulum or embedding them in its membrane.
- - Recent discoveries in structural biology have provided insight into how Sec61 interacts with other proteins and have identified small molecules that can inhibit its function.
- - Some of these molecules can selectively block the transport of specific proteins, suggesting potential strategies for targeting Sec61 in drug development while minimizing harmful effects.
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