PROJECT SUMMARY Nearly 30% of the eukaryotic genome encodes integral membrane proteins, which serve many essential functions as receptors, enzymes, anchors and transporters. Membrane proteins of the cell surface and most intracellular compartments are first assembled at the endoplasmic reticulum (ER). These proteins are cotranslationally targeted to the ER by the signal recognition particle and inserted into the bilayer by the Sec61 complex. In the simplest view, the core Sec61 complex mediates insertion by guiding nascent hydrophobic transmembrane domains (TMDs) into a central, aqueous pore which opens laterally to allow TMD entry into the bilayer. While this model has proven valuable for understanding the basic mechanism of TMD insertion, its application to the biogenesis of physiologic substrates—especially those with multiple TMDs—has been challenging. This challenge arises from the extreme diversity of eukaryotic membrane proteins, which have drastically different topologies and biophysical requirements for insertion, folding, modification and assembly into functional entities. These different steps are coordinated by the `translocon', a poorly defined and dynamic ensemble comprising the Sec61 complex in association with a variety of accessory subunits. The structures, stoichiometry and functions of most of this machinery are poorly understood, and their roles in membrane protein biogenesis are largely unexplored. Understanding how different translocon complexes mediate membrane biogenesis is a fundamental question in cell biology We recently classified a conserved but poorly understood human protein called TMCO1 as a member of a previously unrecognized superfamily of proteins involved in membrane protein biogenesis. Consistent with this assignment, our preliminary data demonstrate that TMCO1 is part of a multi-component assembly that includes the Sec61 complex and ribosomes, and directly link it to a role in the cotranslational insertion, folding and/or assembly of a large group of membrane proteins. Here we build on this conceptual and technical foundation to define how TMCO1 functions in membrane protein biogenesis. In Aim 1, we will globally identify the set of nascent TMCO1 substrates and use these to define the mechanism of TMCO1 action. In Aim 2, we will rigorously analyze the interaction partners of TMCO1 and define the structure of TMCO1-containing ribosome-Sec61 complexes. We will do this using a multi-disciplinary approach that combines biochemical, genetic and structural analyses.