Title: Revealing the inner workings of the EMC, a multifunctional membrane protein biogenesis factor.
2Center for Integrated Protein Science at the Department of Chemistry, Technical University of Munich, Germany
3Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
4Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
5Department of Biology, Whitehead Institute, MIT, Cambridge, United States
7Howard Hughes Medical Institute, Chevy Chase, United States
Membrane proteins rely on a large suite of biogenesis and quality control factors to acquire correct folding and topology within cellular membranes: their misfolding underlies numerous severe human diseases. The ER is a large membrane organelle containing many membrane proteins, which perform diverse tasks from synthesis of lipids to regulation of cellular membrane trafficking. The well-conserved endoplasmic reticulum membrane protein complex (EMC) has emerged as an important player in membrane protein homeostasis. While its role as a post-translational insertase for tail-anchored transmembrane helices has been studied in detail, other more elusive functions in facilitating biogenesis of multi-pass membrane proteins remain to be mechanistically defined. We are taking an interdisciplinary approach to illuminate EMC biochemistry: 1) Cryo-EM structures of yeast and human EMC reveal large soluble domains representing interaction platforms in cytosol and ER lumen; at least four core subunits form two cavities inside the ER membrane differing in lipid accessibility. 2) Broad structure-guided mutagenesis across the EMC, combined with a cell-based functional assay for EMC client stability, uncovered separation of function across this large molecular machine for three different client topologies: tail-anchored, luminal N-terminus and cytosolic N-terminus. Mutants presenting opposing effects on client stability suggest regulated client biogenesis through conformational cycling or lipid binding. 3) A genome-wide CRISPRi screen for factors affecting the stability of the tail-anchored EMC-dependent client squalene synthase uncovered additional ER resident and cytosolic factors which may cooperate with EMC in client biogenesis. We are following up on several of these screen hits to try and understand how they influence EMC client stability. Moreover, we are pursuing cryo-EM structures of EMC with additional interactors, which have emerged from proteomics analyses using endogenously affinity-tagged proteins. Together, our interdisciplinary efforts spanning structural- and cell biology will hopefully lead to a more complete picture of how EMC assists the biogenesis of an extremely broad client range, including several classes of ion channels, GPCRs and lipid biosynthetic enzymes.