An ER translocon for multi-pass membrane protein biogenesis.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Cell membranes are structures that separate the interior of the cell from its environment and determine the cell's shape and the structure of its internal compartments. Nearly 25% of human genes encode transmembrane proteins that span the entire membrane from one side to the other, helping the membrane perform its roles. Transmembrane proteins are synthesized by ribosomes ­ protein-making machines ­ that are on the surface of a cell compartment called the endoplasmic reticulum. As the new protein is made by the ribosome, it enters the endoplasmic reticulum membrane where it folds into the correct shape. This process is best understood for proteins that span the membrane once. Despite decades of work, however, much less is known about how multi-pass proteins that span the membrane multiple times are made. A study from 2017 showed that a protein called TMCO1 is related to a group of proteins involved in making membrane proteins. TMCO1 has been linked to glaucoma, and mutations in it cause cerebrofaciothoracic dysplasia, a human disease characterized by severe intellectual disability, distinctive facial features, and bone abnormalities. McGilvray, Anghel et al. ­ including several of the researchers involved in the 2017 study ­ wanted to determine what TMCO1 does in the cell and begin to understand its role in human disease. McGilvray, Anghel et al. discovered that TMCO1, together with other proteins, is part of a new 'translocon' ­ a group of proteins that transports proteins into the endoplasmic reticulum membrane. Using a combination of biochemical, genetic and structural techniques, McGilvray, Anghel et al. showed that the translocon interacts with ribosomes that are synthesizing multi-pass proteins. The experiments revealed that the translocon is required for the production of a multi-pass protein called EAAT1, and it provides multiple ways for proteins to be inserted into and folded within the membrane. The findings of McGilvray, Anghel et al. reveal a previously unknown cellular machinery which may be involved in the production of hundreds of human multi-pass proteins. This work provides a framework for understanding how these proteins are correctly made in the membrane. Additionally, it suggests that human diseases caused by mutations in TMCO1 result from a defect in the production of multi-pass membrane proteins.

Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum.

Members of the evolutionarily conserved Oxa1/Alb3/YidC family mediate membrane protein biogenesis at the mitochondrial inner membrane, chloroplast thylakoid membrane, and bacterial plasma membrane, respectively. Despite their broad phylogenetic distribution, no Oxa1/Alb3/YidC homologs are known to operate in eukaryotic cells outside the endosymbiotic organelles. Here, we present bioinformatic evidence that the tail-anchored protein insertion factor WRB/Get1, the "endoplasmic reticulum (ER) membrane complex" subunit EMC3, and TMCO1 are ER-resident homologs of the Oxa1/Alb3/YidC family. Topology mapping and co-evolution-based modeling demonstrate that Get1, EMC3, and TMCO1 share a conserved Oxa1-like architecture. Biochemical analysis of human TMCO1, the only homolog not previously linked to membrane protein biogenesis, shows that it associates with the Sec translocon and ribosomes. These findings suggest a specific biochemical function for TMCO1 and define a superfamily of proteins-the "Oxa1 superfamily"-whose shared function is to facilitate membrane protein biogenesis.

A YidC-like Protein in the Archaeal Plasma Membrane.

Cells possess specialized machinery to direct the insertion of membrane proteins into the lipid bilayer. In bacteria, the essential protein YidC inserts certain proteins into the plasma membrane, and eukaryotic orthologs are present in the mitochondrial inner membrane and the chloroplast thylakoid membrane. The existence of homologous insertases in archaea has been proposed based on phylogenetic analysis. However, limited sequence identity, distinct architecture, and the absence of experimental data have made this assignment ambiguous. Here we describe the 3.5-Å crystal structure of an archaeal DUF106 protein from Methanocaldococcus jannaschii (Mj0480), revealing a lipid-exposed hydrophilic surface presented by a conserved YidC-like fold. Functional analysis reveals selective binding of Mj0480 to ribosomes displaying a stalled YidC substrate, and a direct interaction between the buried hydrophilic surface of Mj0480 and the nascent chain. These data provide direct experimental evidence that the archaeal DUF106 proteins are YidC/Oxa1/Alb3-like insertases of the archaeal plasma membrane.