Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed.

A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER.

Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA proteins to the ER, where they are delivered to the insertase, the ER membrane protein complex (EMC). Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA protein from its cytosolic capture by methionine-rich loops to its membrane insertion through a hydrophilic vestibule.

A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum.

Tail-anchored (TA) proteins are an essential class of integral membrane proteins required for many aspects of cellular physiology. TA proteins contain a single carboxy-terminal transmembrane domain that must be post-translationally recognized, guided to, and ultimately inserted into the correct cellular compartment. The majority of TA proteins begin their biogenesis in the endoplasmic reticulum (ER) and utilize two parallel strategies for targeting and insertion: the guided-entry of tail-anchored proteins (GET) and ER-membrane protein complex (EMC) pathways.

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.

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.