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.

Most membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes docked at the translocon, a heterogeneous ensemble of transmembrane factors operating on the nascent chain1,2. How the translocon coordinates the actions of these factors to accommodate its different substrates is not well understood. Here we define the composition, function and assembly of a translocon specialized for multipass membrane protein biogenesis3. This 'multipass translocon' is distinguished by three components that selectively bind the ribosome-Sec61 complex during multipass protein synthesis: the GET- and EMC-like (GEL), protein associated with translocon (PAT) and back of Sec61 (BOS) complexes. Analysis of insertion intermediates reveals how features of the nascent chain trigger multipass translocon assembly. Reconstitution studies demonstrate a role for multipass translocon components in protein topogenesis, and cells lacking these components show reduced multipass protein stability. These results establish the mechanism by which nascent multipass proteins selectively recruit the multipass translocon to facilitate their biogenesis. More broadly, they define the ER translocon as a dynamic assembly whose subunit composition adjusts co-translationally to accommodate the biosynthetic needs of its diverse range of substrates.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

Hundreds of proteins are inserted post-translationally into the endoplasmic reticulum (ER) membrane by a single carboxy-terminal transmembrane domain (TMD). During targeting through the cytosol, the hydrophobic TMD of these tail-anchored (TA) proteins requires constant chaperoning to prevent aggregation or inappropriate interactions. A central component of this targeting system is TRC40, a conserved cytosolic factor that recognizes the TMD of TA proteins and delivers them to the ER for insertion. The mechanism that permits TRC40 to find and capture its TA protein cargos effectively in a highly crowded cytosol is unknown. Here we identify a conserved three-protein complex composed of Bat3, TRC35 and Ubl4A that facilitates TA protein capture by TRC40. This Bat3 complex is recruited to ribosomes synthesizing membrane proteins, interacts with the TMDs of newly released TA proteins, and transfers them to TRC40 for targeting. Depletion of the Bat3 complex allows non-TRC40 factors to compete for TA proteins, explaining their mislocalization in the analogous yeast deletion strains. Thus, the Bat3 complex acts as a TMD-selective chaperone that effectively channels TA proteins to the TRC40 insertion pathway.

Whole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.

We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions.

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.

Patient-reported data can improve quality of healthcare delivery and patient outcomes. Moffitt Cancer Center ("Moffitt") administers the Electronic Patient Questionnaire (EPQ) to collect data on demographics, including sexual orientation and gender identity (SOGI), medical history, cancer risk factors, and quality of life. Here we investigated differences in EPQ completion by demographic and cancer characteristics.

The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.

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.

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane.

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.