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

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

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

Identifying and Characterizing a Functional HIV-1 Reverse Transcriptase-binding Site on Integrase*

Integrase (IN) from human immunodeficiency virus, type 1 (HIV-1) exerts pleiotropic effects in the viral replication cycle. Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription. IN and reverse transcriptase (RT) interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN-RT interaction. An IN K258A substitution that disrupts reverse transcription in infected cells is located at the putative RT-binding surface, and we found that this substitution substantially weakens IN CTD-RT interactions. We also identified two additional IN amino acid substitutions located at the putative RT-binding surface (W243E and V250E) that significantly impair viral replication in tissue culture. These results strengthen the notion that IN-RT interactions are biologically relevant during HIV-1 replication and also provide insights into this interaction at the molecular level