The proper functioning of any living cell requires that approximately half of all proteins synthesized in the cytosol must be translocated across (in the case of exported proteins) or inserted into (in the case of membrane proteins) a cell membrane. Protein translocation at the membrane occurs through a proteinaceous channel, termed the translocon. The core of the translocon is a heterotrimeric integral membrane protein complex (SecY, SecE and SecG in eubacteria) and is conserved in all kingdoms of life. Little is known about the structure and function of the additional components of the holo-translocation machinery, SecD, SecF and YidC, which are essential for eubacteria. In spite of their central role, the function of these subunits is entirely unresolved to date. This is to a large part due to the lack of a purified, reconstituted SecYEG-DF-YidC holo-translocon, and the absence of any structural data on this large transmembrane multiprotein complex. Using a new recombineering-based vector system for expression of multi-protein complexes in E. coli, we now for the first time successfully over-expressed and purified the SecYEG-DF-YidC holo-translocon. With this purified holo-translocon in hand, we are now in a unique position to carry out a thorough functional and structural characterization of this vitally important membrane protein complex. Our project will be divided in five complementary parts: (1) We will solve the structure of the holo-translocon by cryo-electron microscopy and single particle analysis. (2) We will study co-translational translocation by solving the structure of the holo-translocon complex with a translating ribosome by cryo-electron microscopy and single particle analysis. Using the existing structures of the ribosome, SecYEG and the predicted homology of SecDF to AcrB, we will build a quasi-atomic model of the holo-translocon. (3) We will thoroughly assay the molecular level function of the holo-translocon in protein translocation, in a collaborative setup with Prof. Ian Collinson, Bristol, UK. The functional modulation of SecA-driven post-translational translocation by SecDF and the effect of the proton motive force will be investigated. (4) The stoichiometry of the subunits in the holo-translocon complex is an entirely unresolved issue to date. We will analyze the stoichiometry and the architecture of the holo-translocon by mass spectrometry, in collaboration with Prof. Carol V. Robinson, Cambridge, UK. (5) We will perform crystallization experiments of the holo-translocon for analysis by 2D electron crystallography (with Prof. Ian Collinson, Bristol) and for 3D X-ray crystallography by using the high-throughput crystallization facilities that are part of the Partnership in Structural Biology (PSB) here in Grenoble. A molecular understanding of protein export and membrane protein integration and folding will not only provide fundamental insight into a paramountly important biological process, but may also contribute significantly to the future design of novel, and urgently needed antibiotics.