Structural basis for membrane insertion by the human ER membrane protein complex

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms by cryo–electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer

Structural basis for membrane insertion by the human ER membrane protein complex

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms by cryo–electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer

Regulated assembly of the ER membrane protein complex

The assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome

WNK1 is an assembly factor for the human ER membrane protein complex

The assembly of nascent proteins into multi-subunit complexes is a tightly regulated process that must occur at high fidelity to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble cytosolic subunits to function. Here, we show that the kinase with no lysine 1 (WNK1), known for its role in hypertension and neuropathy, functions as an assembly factor for the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2–8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and directly competes for binding of E3 ubiquitin ligases, permitting assembly. Depletion of WNK1 thus destabilizes both the EMC and its membrane protein clients. This work describes an unexpected role for WNK1 in protein biogenesis and defines the general requirements of an assembly factor that will apply across the proteome

Structure and Mechanism of the Human ER Membrane Protein Complex

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

Architecture of the linker-scaffold in the nuclear pore

Quantitative docking of crystal and single particle cryo-EM structures into low resolution human and S. cerevisiae cryo-ET and X. laevis cryo-EM maps of the nuclear pore complex.Related Publication: Architecture of the linker-scaffold in the nuclear pore Petrovic, Stefan Caltech Samanta, Dipanjan Caltech Perriches, Thibaud Caltech Bley, Christopher Caltech Thierbach, Karsten Caltech Brown, Bonnie Caltech Nie, Si Caltech Mobbs, George Caltech Stevens, Taylor Caltech Liu, Xiaoyu Caltech Tomaleri, Giovani Pinton Caltech Schaus, Lucas Caltech Hoelz, Andre Caltech Science 2022-06-10 https://doi.org/10.1126/science.abm9798 en