Structure and Mechanism of the Human ER Membrane Protein Complex

Citation

Pinton Tomaleri, Giovani (2023) Structure and Mechanism of the Human ER Membrane Protein Complex. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/e85y-re86. https://resolver.caltech.edu/CaltechTHESIS:06012023-220838845

Abstract

The successful synthesis, targeting, insertion, folding, and assembly of membrane proteins into designated membranes is a crucial process in cell biology. Recent research has shed valuable light on this process through the discovery of the endoplasmic reticulum (ER) membrane protein complex (EMC) and its role in membrane protein biogenesis and quality control. As part of my Ph.D. research in the Voorhees lab, I collaborated with esteemed scientists to investigate the EMC in atomic detail. Described in this thesis is the mechanistic basis of EMC function in membrane protein biogenesis, including recent insights into its broader role beyond its well-defined insertase function. First, we determined the structure of the human EMC using single-particle cryo-electron microscopy (cryo-EM). The structure revealed that it utilizes a mechanism similar to other protein-conducting channels, which involves membrane thinning and polar intramembrane residues to transport substrate transmembrane domains from the cytosol into the membrane. The EMC structure provided the foundation for the subsequent rigorous analysis of its role in membrane protein biogenesis. Through this work, we demonstrate the molecular mechanisms involved in the EMC-dependent path a membrane protein takes, from its initial cytosolic capture by methionine-rich loops of the EMC to its eventual membrane insertion via a hydrophilic vestibule. We further show that specific polar intramembrane residues on the EMC serve as an ER “selectivity filter” which uses charge-repulsion properties to reject mis-targeted mitochondrial membrane proteins and maintain organelle integrity. We also demonstrate that the EMC ensures that transmembrane-spanning substrates adopt the correct topology by promoting the “positive-inside” rule, which states that positively and negatively charged amino acids localize to the interior (cytoplasmic) and exterior (non-cytoplasmic) sides of membranes, respectively. Finally, our studies suggest that the EMC has a broader role beyond its well-defined insertase function. Specifically, we found that the EMC physically binds to other factors involved in membrane protein biogenesis, providing a shared interaction surface that acts as a hub to integrate signals from other pathways. Using a combination of structural and functional approaches, we identified the EMC’s interaction with Nodal modulator (NOMO) complex, which is part of the multipass translocon complex and facilitate membrane protein biogenesis. Together, these results define and expand the model for membrane protein biogenesis at the ER membrane by the EMC and highlight the complex interplay between different factors in this important process. Together, these results define and expand the model for membrane protein biogenesis at the ER membrane by the EMC and highlight the complex interplay between different factors in this important process.

Thesis Files