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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. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells., Competing Interests: Declaration of interests R.M.V. is a consultant and equity holder, and G.P.T. is a current employee, of Gate Bioscience., (Copyright © 2024. Published by Elsevier Inc.)

α-Helical integral membrane proteins comprise approximately 25% of the proteome in all organisms. The membrane proteome is highly diverse, varying in the number, topology, spacing and properties of transmembrane domains. This diversity imposes different constraints on the insertion of different regions of a membrane protein into the lipid bilayer. Here, we present a cohesive framework to explain membrane protein biogenesis, in which different parts of a nascent substrate are triaged between Oxa1 and SecY family members for insertion. In this model, Oxa1 family proteins insert transmembrane domains flanked by short translocated segments, whereas the SecY channel is required for insertion of transmembrane domains flanked by long translocated segments. Our unifying model rationalizes evolutionary, genetic, biochemical and structural data across organisms and provides a foundation for future mechanistic studies of membrane protein biogenesis., (© 2024. Springer Nature America, Inc.)

Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome., (© 2024 Gemmer et al.)

Membrane protein insertion into and translocation across the bacterial cytoplasmic membrane are essential processes facilitated by the Sec translocon. Membrane insertion occurs co-translationally whereby the ribosome nascent chain is targeted to the translocon via signal recognition particle and its receptor FtsY. The phospholipid dependence of membrane protein insertion has remained mostly unknown. Here we assessed in vitro the dependence of the SecA independent insertion of the mannitol permease MtlA into the membrane on the main phospholipid species present in Escherichia coli. We observed that insertion depends on the presence of phosphatidylglycerol and is due to the anionic nature of the polar headgroup, while insertion is stimulated by the zwitterionic phosphatidylethanolamine. We found an optimal insertion efficiency at about 30 mol% DOPG and 50 mol% DOPE which approaches the bulk membrane phospholipid composition of E. coli., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Arnold J.M. Driessen reports financial support was provided by Dutch Research Council., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The epithelial cell-adhesion molecule (EpCAM) is hyperglycosylated in carcinoma tissue and the oncogenic function of EpCAM primarily depends on the degree of glycosylation. Inhibiting EpCAM glycosylation is expected to have an inhibitory effect on cancer. We analyzed the relationship of BAP31 with 84 kinds of tumor-associated antigens and found that BAP31 is positively correlated with the protein level of EpCAM. Triple mutations of EpCAM N76/111/198A, which are no longer modified by glycosylation, were constructed to determine whether BAP31 has an effect on the glycosylation of EpCAM. Plasmids containing different C-termini of BAP31 were constructed to identify the regions of BAP31 that affects EpCAM glycosylation. Antibodies against BAP31 (165-205) were screened from a human phage single-domain antibody library and the effect of the antibody (VH-F12) on EpCAM glycosylation and anticancer was investigated. BAP31 increases protein levels of EpCAM by promoting its glycosylation. The amino acid region from 165 to 205 in BAP31 plays an important role in regulating the glycosylation of EpCAM. The antibody VH-F12 significantly inhibited glycosylation of EpCAM which, subsequently, reduced the adhesion of gastric cancer cells, inducing cytotoxic autophagy, inhibiting the AKT-PI3K-mTOR signaling pathway, and, finally, resulting in proliferation inhibition both in vitro and in vivo. Finally, we clarified that BAP31 plays a key role in promoting N-glycosylation of EpCAM by affecting the Sec61 translocation channels. Altogether, these data implied that BAP31 regulates the N-glycosylation of EpCAM and may represent a potential therapeutic target for cancer therapy.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis., (© 2023. The Author(s).)

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

YidC is a member of the YidC/Oxa1/Alb3 protein family that is crucial for membrane protein biogenesis in the bacterial plasma membrane. While YidC facilitates the folding and complex assembly of membrane proteins along with the Sec translocon, it also functions as a Sec-independent membrane protein insertase in the YidC-only pathway. However, little is known about how membrane proteins are recognized and sorted by these pathways, especially in Gram-positive bacteria, for which only a small number of YidC substrates have been identified to date. In this study, we aimed to identify Bacillus subtilis membrane proteins whose membrane insertion depends on SpoIIIJ, the primary YidC homolog in B. subtilis. We took advantage of the translation arrest sequence of MifM, which can monitor YidC-dependent membrane insertion. Our systematic screening identified eight membrane proteins as candidate SpoIIIJ substrates. Results of our genetic study also suggest that the conserved arginine in the hydrophilic groove of SpoIIIJ is crucial for the membrane insertion of the substrates identified here. However, in contrast to MifM, a previously identified YidC substrate, the importance of the negatively charged residue on the substrates for membrane insertion varied depending on the substrate. These results suggest that B. subtilis YidC uses substrate-specific interactions to facilitate membrane insertion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Multipass membrane proteins contain two or more α-helical transmembrane domains (TMDs) that span the lipid bilayer. They are inserted cotranslationally into the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum membrane. The Sec61 complex (SecY complex in prokaryotes) provides a ribosome docking site, houses a channel across the membrane, and contains a lateral gate that opens toward the lipid bilayer. Model multipass proteins can be stitched into the membrane by iteratively using Sec61's lateral gate for TMD insertion and its central pore for translocation of flanking domains. Native multipass proteins, with their diverse TMDs and complex topologies, often also rely on members of the Oxa1 family of translocation factors, the PAT complex chaperone, and other poorly understood factors. Here, we discuss the mechanisms of TMD insertion, highlight the limitations of an iterative insertion model, and propose a new hypothesis for multipass membrane protein biogenesis based on recent findings., (Copyright © 2023 Cold Spring Harbor Laboratory Press; all rights reserved.)

Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Most membrane proteins use their first transmembrane domain, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology is not understood. Using site-specific crosslinking, we detect a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. These results suggest that EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis., Competing Interests: Declaration of interests R.S.H. is a member of the advisory board for Molecular Cell., (Copyright © 2023 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)

The endoplasmic reticulum (ER) is a major site for protein synthesis, folding, and maturation in eukaryotic cells, responsible for production of secretory proteins and most integral membrane proteins. The universally conserved protein-conducting channel Sec61 complex mediates core steps in these processes by translocating hydrophilic polypeptide segments of client proteins across the ER membrane and integrating hydrophobic transmembrane segments into the membrane. The Sec61 complex associates with several other molecular machines and enzymes to enable substrate engagement with the channel and coordination of protein translocation with translation, protein folding, and/or post-translational modifications. Recent cryo-electron microscopy and functional studies of these translocon complexes have greatly advanced our mechanistic understanding of Sec61-dependent protein biogenesis at the ER. Here, we will review the current models for how the Sec61 channel performs its functions in coordination with partner complexes., (Copyright © 2023 Cold Spring Harbor Laboratory Press; all rights reserved.)

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecA N protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts., Competing Interests: Declaration of Competing Interest We declare that we have no conflict of interest to this work., (Copyright © 2022. Published by Elsevier B.V.)

More than 30% of eukaryotic proteins contain domains that must translocate across or integrate into the endoplasmic reticulum (ER) membrane. With few exceptions, protein translocation and transmembrane domain integration at the ER require the conserved Sec61 translocon. Decades of studies have established a clear mechanistic model for how the Sec61 translocon functions. The biosynthesis of distinct subsets of proteins at the ER also involves accessory factors that interact with the Sec61 translocon and translocating nascent proteins. However, assigning specific functions to many translocon accessory factors has been a persistent challenge in the field. This Perspective discusses recent insights into mechanisms that promote protein biosynthesis at the ER through accessory factors that directly regulate the Sec61 translocon or chaperone nascent proteins within the ER membrane. These translocon accessory factor functions, and more still to be discovered, are essential for producing a diverse and high-fidelity proteome at the ER.

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 chain 1,2 . 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 biogenesis 3 . 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., (© 2022. The Author(s).)

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways., (© 2022. The Author(s).)

The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB's two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Over 100 glycosylphosphatidylinositol-anchored proteins (GPI-APs) are encoded in the mammalian genome. It is not well understood how these proteins are targeted and translocated to the endoplasmic reticulum (ER). Here, we reveal that many GPI-APs, such as CD59, CD55, and CD109, utilize human SND2 (hSND2)-dependent ER targeting machinery. We also found that signal recognition particle receptors seem to cooperate with hSND2 to target GPI-APs to the ER. Both the N-terminal signal sequence and C-terminal GPI attachment signal of GPI-APs contribute to ER targeting via the hSND2-dependent pathway. Particularly, the hydrophobicity of the C-terminal GPI attachment signal acts as the determinant of hSND2 dependency. Our results explain the route and mechanism of the ER targeting of GPI-APs in mammalian cells., (© 2021 Federation of European Biochemical Societies.)

A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins., Competing Interests: Conflict of interest—The authors declare that no competing interests exits., (© 2020 Sun and Mariappan.)

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [ 35 S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Hannigan et al.)

Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries., Competing Interests: The authors have declared that no competing interests exist.

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

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., Competing Interests: PM, SA, AS, FZ, MT, JF, HH, AB, RK No competing interests declared, (© 2020, McGilvray et al.)

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo -generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae , a few proteins devoid of a signal peptide are still translocated into the ER and then N -glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain ( ste24 Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N -glycosylated in the ER in the ste24 Δ strain, we identified the transcription factor Rme1 as a protein that is partially N -glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Hosomi et al.)

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., Competing Interests: JO, BP, YY, SJ, AW, DM, RK No competing interests declared, EM, RH Reviewing editor, eLife, (© 2020, O'Donnell et al.)

Membrane protein folding studies lag behind those of water-soluble proteins due to immense difficulties of experimental study, resulting from the need to provide a hydrophobic lipid-bilayer environment when investigated in vitro. A sound understanding of folding mechanisms is important for membrane proteins as they contribute to a third of the proteome and are frequently associated with disease when mutated and/or misfolded. Membrane proteins largely consist of α-helical, hydrophobic transmembrane domains, which insert into the membrane, often using the SecYEG/Sec61 translocase system. This mini-review highlights recent advances in techniques that can further our understanding of co-translational folding and notably, the structure and insertion of nascent chains as they emerge from translating ribosomes. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins., (Copyright © 2019. Published by Elsevier B.V.)

Various growth factors and full-length cell surface receptors such as EGFR are translocated from the cell surface to the nucleoplasm, baffling cell biologists to the mechanisms and functions of this process. Elevated levels of nuclear EGFR correlate with poor prognosis in various cancers. In recent years, nuclear EGFR has been implicated in regulating gene transcription, cell proliferation and DNA damage repair. Different models have been proposed to explain how the receptors are transported into the nucleus. However, a clear consensus has yet to be reached. Recently, we described the nuclear envelope associated endosomes (NAE) pathway, which delivers EGFR from the cell surface to the nucleus. This pathway involves transport, docking and fusion of NAEs with the outer membrane of the nuclear envelope. EGFR is then presumed to be transported through the nuclear pore complex, extracted from membranes and solubilised. The SUN1/2 nuclear envelope proteins, Importin-beta, nuclear pore complex proteins and the Sec61 translocon have been implicated in the process. While this framework can explain the cell surface to nucleus traffic of EGFR and other cell surface receptors, it raises several questions that we consider in this review, together with implications for health and disease., Competing Interests: The authors declare no conflict of interest

The membrane of the endoplasmic reticulum (ER) in human cells harbors the protein translocon, which facilitates membrane insertion and translocation of almost every newly synthesized polypeptide targeted to organelles of the secretory pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins, which are associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, Sec61 channel opening and closing, and modification of precursor polypeptides in transit through the Sec61 complex. Recently, cryoelectron tomography of translocons in native ER membranes has given unprecedented insights into the architecture and dynamics of the native, ribosome-associated translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion or translocation of newly synthesized polypeptides as well as the possible roles of the Sec61 channel as a passive ER calcium leak channel and regulator of ATP/ADP exchange between cytosol and ER.

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the β-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F 1 F O adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid-bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

About 30% of all bacterial proteins execute their function outside of the cytosol and have to be transported into or across the cytoplasmic membrane. Bacteria use multiple protein transport systems in parallel, but the majority of proteins engage two distinct targeting systems. One is the co-translational targeting by two universally conserved GTPases, the signal recognition particle (SRP) and its receptor FtsY, which deliver inner membrane proteins to either the SecYEG translocon or the YidC insertase for membrane insertion. The other targeting system depends on the ATPase SecA, which targets secretory proteins, i.e. periplasmic and outer membrane proteins, to SecYEG for their subsequent ATP-dependent translocation. While SRP selects its substrates already very early during their synthesis, the recognition of secretory proteins by SecA is believed to occur primarily after translation termination, i.e. post-translationally. In this review we highlight recent progress on how SRP recognizes its substrates at the ribosome and how the fidelity of the targeting reaction to SecYEG is maintained. We furthermore discuss similarities and differences in the SRP-dependent targeting to either SecYEG or YidC and summarize recent results that suggest that some membrane proteins are co-translationally targeted by SecA.

The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise heterogeneously in the membrane of the bacterium Staphylococcus aureus, we have analysed the localisation of other key membrane proteins. A range of protein fusions were constructed and used in conjunction with quantitative image analysis. Enzymes involved in phospholipid biosynthesis as well as the lipid raft marker FloT exhibited a heterogeneous localisation pattern. However, the secretion associated SecY protein, was more homogeneously distributed in the membrane. A FRET-based system also identified novel colocalisation between phospholipid biosynthesis enzymes and the respiratory protein CydB revealing a likely larger network of partners. PlsY localisation was found to be dose dependent but not to be affected by membrane lipid composition. Disruption of the activity of the essential cell division organiser FtsZ, using the inhibitor PC190723 led to loss of PlsY localisation, revealing a link to cell division and a possible role for FtsZ in functions not strictly associated with septum formation.

In metazoan organisms, the STT3A isoform of the oligosaccharyltransferase is localized adjacent to the protein translocation channel to catalyze co-translational N-linked glycosylation of proteins in the endoplasmic reticulum. The mechanism responsible for the interaction between the STT3A complex and the translocation channel has not been addressed. Using genetically modified human cells that are deficient in DC2 or KCP2 proteins, we show that loss of DC2 causes a defect in co-translational N-glycosylation of proteins that mimics an STT3A -/- phenotype. Biochemical analysis showed that DC2 and KCP2 are responsible for mediating the interaction between the protein translocation channel and the STT3A complex. Importantly, DC2- and KCP2-deficient STT3A complexes are stable and enzymatically active. Deletion mutagenesis revealed that a conserved motif in the C-terminal tail of DC2 is critical for assembly into the STT3A complex, whereas the lumenal loop and the N-terminal cytoplasmic segment are necessary for the functional interaction between the STT3A and Sec61 complexes., (© 2017 Shrimal et al.)

Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the "Helical Scaffold" Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.

The majority of multi-spanning membrane proteins are co-translationally inserted into the bilayer by the Sec pathway. An important subset of membrane proteins have globular, cofactor-containing extracytoplasmic domains requiring the dual action of the co-translational Sec and post-translational Tat pathways for integration. Here, we identify further unexplored families of membrane proteins that are dual Sec-Tat-targeted. We establish that a predicted heme-molybdenum cofactor-containing protein, and a complex polyferredoxin, each require the concerted action of two translocases for their assembly. We determine that the mechanism of handover from Sec to Tat pathway requires the relatively low hydrophobicity of the Tat-dependent transmembrane domain. This, coupled with the presence of C-terminal positive charges, results in abortive insertion of this transmembrane domain by the Sec pathway and its subsequent release at the cytoplasmic side of the membrane. Together, our data points to a simple unifying mechanism governing the assembly of dual targeted membrane proteins.

Some modified glucagon-like-peptide-1 (GLP-1) analogs are highly important for treating type 2 diabetes. Here we investigated whether GLP-1 analogs expressed in Lactococcus lactis could be substrates for modification and export by the nisin dehydratase and transporter enzyme. Subsequently we introduced a lysinoalanine by coupling a formed dehydroalanine with a lysine and investigated the structure and activity of the formed lysinoalanine-bridged GLP-1 analog. Our data show: (i) GLP-1 fused to the nisin leader peptide is very well exported via the nisin transporter NisT, (ii) production of leader-GLP-1 via NisT is higher than via the SEC system, (iii) leader-GLP-1 exported via NisT was more efficiently dehydrated by the nisin dehydratase NisB than when exported via the SEC system, (iv) individual serines and threonines in GLP-1 are dehydrated by NisB to a significantly different extent, (v) an introduced Ser30 is well dehydrated and can be coupled to Lys34 to form a lysinoalanine-bridged GLP-1 analog, (vi) a lysinoalanine(30-34) variant's conformation shifts in the presence of 25% trifluoroethanol towards a higher alpha helix content than observed for wild type GLP-1 under identical condition, (vii) a lysinoalanine(30-34) GLP-1 variant has retained significant activity. Taken together the data extend knowledge on the substrate specificities of NisT and NisB and their combined activity relative to export via the Sec system, and demonstrate that introducing a lysinoalanine bridge is an option for modifying therapeutic peptides., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans , the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression that are characteristic of this disease result from the action of mycolactone, which targets the Sec61 complex and inhibits the co-translational translocation of secretory proteins into the endoplasmic reticulum. In this study, we investigate the effect of mycolactone on the Sec61-dependent biogenesis of different classes of transmembrane protein (TMP). Our data suggest that the effect of mycolactone on TMP biogenesis depends on how the nascent chain initially engages the Sec61 complex. For example, the translocation of TMP lumenal domains driven by an N-terminal cleavable signal sequence is efficiently inhibited by mycolactone. In contrast, the effect of mycolactone on protein translocation that is driven solely by a non-cleavable signal anchor/transmembrane domain depends on which flanking region is translocated. For example, while translocation of the region N-terminal to a signal anchor/transmembrane domain is refractive to mycolactone, C-terminal translocation is efficiently inhibited. Our findings highlight the diversity of Sec61-dependent translocation and provide a molecular basis for understanding the effect of mycolactone on the biogenesis of different TMPs., (© 2017. Published by The Company of Biologists Ltd.)

Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions.

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase., (© 2016 Kida et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)