Using Functional Genomics to Characterize Biogenesis and Quality Control Pathways in the Mammalian ER

Citation

Page, Katharine-Rose (2024) Using Functional Genomics to Characterize Biogenesis and Quality Control Pathways in the Mammalian ER. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/4c7k-yw62. https://resolver.caltech.edu/CaltechTHESIS:11062023-233057820

Abstract

Cells are tasked with ensuring the proper synthesis, localization, and insertion of membrane proteins at the mammalian endoplasmic reticulum (ER). Recent advances have shown that the insertion of transmembrane domains into the ER and the translocation of their associated soluble domains across the ER can be facilitated by members of the Oxa1 superfamily of insertases. The ER membrane protein complex (EMC) contains an Oxa1 insertase that facilitates the co-translational insertion of the first TMD of Nexo proteins, which have their N-terminal soluble domains localized in the ER lumen or extracellular space. Additionally, recent work has described the multipass translocon, a supercomplex at the Sec61 translocation channel that facilitates insertion of multipass membrane proteins. During my PhD and in collaboration with other scientists in the Voorhees lab, I elucidated how the EMC cooperates with the multipass translocon to facilitate biogenesis of multipass membrane proteins. We took a systematic approach, applying a combination of functional genomics, biochemistry, structural biology, and mechanistic cell biology to understand how the biophysical properties of the TMDs and the intervening soluble domains of multipass membrane proteins influence their path into the ER bilayer. We show that the EMC is epistatic with members of the multipass translocon, including the BOS, GEL, and PAT complexes. We structurally characterize the EMC•BOS holocomplex, showing that these complexes directly interact, and that this interaction is mutually exclusive to the interaction of BOS and Sec61. Further, we demonstrate that Nexo proteins that contain a net positive charge in their N-terminal soluble domain are difficult for the EMC to insert and thus also rely on Sec61 or TMCO1, the Oxa1 insertase of the GEL complex, for insertion. We overturn the prevailing model for multipass membrane protein insertion and show that how this diverse class of membrane proteins utilizes the suite of ER biogenesis machinery depends on their distinct biophysical properties. In addition to biogenesis, during my PhD I also studied how the cell surveils multi-subunit complex assembly. I focused on a model ER-resident and obligate complex. These subunits are unstable and degraded in the absence of their binding partner, but how they are recognized for degradation is unknown. I used unbiased functional genomics approaches to identify the quality control components that regulate orphan subunit degradation in the cell. Further, I used proteomics to identify endogenous substrates of this particular ERAD pathway. This work expands upon our understanding of how multi-subunit complexes are regulated by machinery in the cell.

Thesis Files