We are interested in the essential process of protein transport. Indeed, about 30% of proteins that are synthesized in the cytosol will need to cross membranes in order to reach their final destination. As membranes are intrinsically impermeable, the question arises: “How does membrane passage of proteins occur? ” In short, it is an energy-dependent process that is mediated by membrane protein complexes called translocases. In the lab, we study the structure and function of the essential and universally conserved system termed the Sec translocon. This nanomachine catalyzes the transport of hundreds of different polypeptide substrates. We analyze the Sec translocon into a functional in vitro translocation system to understand the mechanism at the molecular level.