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Structure and Function of the ER-Membrane Complex 10.17863/CAM.47901 Phillips, Ben Miller, Elizabeth The ER-Membrane Complex (EMC) is a highly conserved heterodecameric membrane protein complex, found in the Endoplasmic Reticulum (ER) membrane. The EMC has been implicated in a wide range of processes including viral reproduction, inter-organelle phospholipid transfer, cholesterol biosynthesis and the biogenesis of multi-pass membrane proteins. Although the complex was formally identified nearly 10 years ago, direct biochemical evidence for functional roles is sparse. Only in the last two years have mechanistic roles been characterised in the insertion of tail-anchored proteins and in defining the topology of the first transmembrane helix in GPCRs. In this thesis I investigate the role of the EMC in the biogenesis of misfolded ABC transporters Yor1$\Delta$FRT and the homologous CFTR$\Delta$F. I expand on previous work on Yor1$\Delta$FRT to reveal that loss of EMC function sensitises cells to expression of a misfolded membrane protein. The biogenesis defect can be re-capitulated by interfering with canonical membrane protein insertion machinery and thus is not specific to $\textit{emc}$$\Delta$ mutants. By studying the $\textit{emc}$$\Delta$ sensitised state I conclude that Yor1$\Delta$FRT is degraded co-translationally. I describe this co-translational folding-driven degradation for the first time. I find that the quality control process utilises machinery shared with cytoplasmic ribosome associated quality control (RQC). I proceed reproduce my observations with parallel experiments for CFTR$\Delta$F. In order to develop our mechanistic understanding of the EMC I set out to solve the structure of the yeast EMC. Structural characterisation via cryo-electron microscopy (cryo-EM) yielded a 6.7Å resolution map that revealed the overall architecture of the complex and multiple features that had not been predicted from previous studies. An appendix includes a 3.5Å resolution cryo-EM map of the Rab GTP exchange factor TRAPPIII bound to its cognate Rab Ypt1. University of Cambridge 2020-06-17 Thesis Doctoral Doctor of Philosophy (PhD) en Medical Research Council (MRC); Boehringer Ingelheim Fonds (BIF) https://www.repository.cam.ac.uk/handle/1810/300825 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3b2b50fb-67f5-4468-8a24-295d4cfccdae/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/be0a02d8-c1cd-49c6-a0af-10bc97af4991/download cc762fe18c88104aebfd61fdfa5c7c02 https://www.rioxx.net/licenses/all-rights-reserved/ Structural Biology Yeast Genetics Biochemistry Biology Membrane Proteins Membrane Biology