The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

Partially inserted nascent chain unzips the lateral gate of the Sec translocon.

The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.

Structure of the post-translational protein translocation machinery of the ER membrane.

Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane1,2. Proteins with highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP)3,4 and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally5,6. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62-Sec63 complex7-9, and substrates are moved through the channel by the luminal BiP ATPase9. How the Sec62-Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel's plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp7010. Our structure shows how the Sec61 channel is activated for post-translational protein translocation.

Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.

Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration.

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 µs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency.