More than 30% of the proteins in all living cells must be transported across or integrated into a membrane, and many of the factors that promote protein insertion are essential for cell viability. Bacteria use two pathways to promote protein insertion into the plasma membrane. The Sec machinery is responsible for inserting the majority of the proteins into the membrane after targeting by the SRP/FtsY components. However, there is also a class of proteins that insert independent of the Sec machinery. These proteins require a novel protein called YidC, which was discovered in the year 2000. YidC is essential for bacterial cell growth, and is evolutionary conserved with homologs in mitochondria and chloroplasts. YidC also works with the Sec machinery to mediate membrane protein insertion. For Sec-dependent proteins, YidC has been proposed to play a role in the lateral transfer of membrane proteins into the lipid bilayer and to function as an assembly site to promote folding of transmembrane domains of membrane proteins. The goal of this research is to understand the exact role of YidC in membrane protein biogenesis. We plan to pursue the following specific aims: (1) Determine the substrate specificity of YidC; (2) Identify the substrate binding region and the organization of the transmembrane domains within the YidC protein; (3) Determine the relationship between YidC, SecYEG, ribosome, SRP/FtsY, and the proteins involved in membrane protein integration; and (4) Determine the function of YidC in membrane protein biogenesis. These studies should impact the field of bacterial membrane protein biogenesis and advance the knowledge of analogous pathways in eukaryotic cells.