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Title: Structure and function of the ER-Membrane Complex
Author: Phillips, Ben
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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The ER-Membrane Complex (EMC) is a highly conserved heterodecameric membrane protein complex, found in the Endoplasmic Reticulum (ER) across almost all branches of eukaryotic life. 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, almost all of the information available that informs a function for the complex comes from indirect high-throughput genetic screens. It was only in 2017 that the first mechanistic study demonstrated that the mammalian EMC can act as a membrane protein insertase for weakly hydrophobic tail-anchored (TA) proteins. Soon after, a second study revealed that the mammalian EMC is required for the determination of topology of the first transmembrane domain (TMD) of a range of G-Protein coupled receptors (GPCRs). In my PhD I set out to characterise the role of the yeast EMC in the biogenesis of a model misfolded membrane protein called Yor1ΔF. Yor1 is broadly homologous to the disease related CFTR transporter and Yor1ΔF replicates the misfolding found in the common disease causing CFTR allele CFTRΔF. Previous work in the Miller lab identified the EMC genes as essential for the biogenesis of Yor1ΔF and in this thesis I expand on that work to reveal that the loss of EMC function sensitises cells to expression of misfolding membrane proteins. By manipulating the emcΔ sensitised state I conclude that the misfolded protein is degraded co-translationally. I describe this co-translational degradation for the first time for a membrane protein and note that it represents the first observation of folding-driven co-translational quality control. I further demonstrate that this process likely utilises similar machinery and mechanisms to analogous cytoplasmic ribosome associated quality control (RQC). I show that this phenomenon can be re-capitulated by interfering with canonical membrane protein insertion machinery and thus is not specific to emc mutants. Thus I describe a novel form of folding-driven co-translational quality control at the ER: ER-RQC. Subsequent to identifying ER-RQC in S. cerevisiae I designed a flow-cytometry based assay to asses whether the role of the EMC is conserved in CFTR biogenesis. Through a variety of genetic and pharmacological manipulations I demonstrate that CFTRΔF is also dependent on the EMC for biogenesis. In order to further our mechanistic understanding of the EMC, and in light of recent developments in the biochemical study of the complex, I set out to solve the structure of the yeast EMC. Initial attempt to purify the yeast EMC were unsuccessful, but a revised approach and optimised purification strategy enabled purification of the human EMC. Structural characterisation via cryo-electron microscopy (cryo-EM) yielded a 6.7Å resolution map that revealed the overall architecture of the complex. The map reveals structural features that had not been previously predicted from sequence analysis and, through incorporation of bioinformatic constraints, it is possible to propose a plausible model for the function of the EMC in membrane protein biogenesis. An appendix includes a 3.5Å resolution cryo-EM map of the Rab GTP exchange factor TRAPPIII bound to its cognate Rab Ypt1. This map is from S. cerevisisae and was solved in collaboration with the Fromme lab at Cornell University (USA).
Supervisor: Miller, Elizabeth Sponsor: Medical Research Council (MRC) ; Boehringer Ingelheim Fonds (BIF)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Structural Biology ; Yeast Genetics ; Biochemistry ; Biology ; Membrane Proteins ; Membrane Biology