The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. One aspect of this proposal is to clarify how proteins are translocated co- or post-translationally from the cytosol across the bacterial plasma membrane. We have determined X-ray and electron-microscopy (EM) structures of the protein-conducting SecY channel, with and without a ribosome-nascent chain complex or the ATPase SecA, and used biochemical experiments to study translocation, providing the basis for the present proposal. In eukaryotes, there is a retro-translocation pathway, called ERAD (for ER associated degradation). We have used purified S. cerevisiae components to reconstitute key steps in ERAD-L used by luminal ER proteins, paving the way for mechanistic studies. Here, we will address central questions in protein translocation: 1. how are proteins cotranslationally translocated? Based on new methods to purify translocation intermediates generated in intact E. coli cells, we will use single- particle cryo-EM to determine how the active channel binds to the ribosome, how the signal sequence binds to the SecY channel, and how the channel opens during translocation. 2. What is the mechanism of posttranslational translocation in bacteria? We will use single-molecule and optical tweezers experiments to test a recently proposed push and slide model in which SecA, upon ATP-binding, pushes a subset of amino acids into the SecY channel and allows passive sliding of the polypeptide in its ADP-bound state. We will use X-ray crystallography to determine how a signal sequence of a post-translational substrate binds to the SecY channel. 3. What is the molecular mechanism of retro-translocation in ERAD-L? We will use in vitro systems with purified ERAD components to determine how proteins are retro-translocated and extracted from the membrane. We will reproduce the substrate specificity observed in wild type cells, analyze the mechanism of individual ERAD-L steps, and determine the specific functions of ERAD components. In vivo experiments in S. cerevisiae will be used to test results obtained in vitro. The mechanism of protein translocation is of great medical importance. Drugs that inhibit signal sequence binding can be used for therapeutic intervention in chronic inflammatory diseases. A large number of diseases, including cystic fibrosis and a1-antitrypsin deficiency, are caused by mutations that result in the misfolding of ER proteins and their subsequent degradation in the cytosol. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.