A unifying model for membrane protein biogenesis.

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.

Getting membrane proteins into shape.

In this issue, Ji et al.1 show how a multipass membrane protein that initially inserts into the endoplasmic reticulum in a mostly inverted topology is post-translationally dislocated, re-inserted, and folded with the help of ATP13A1, a P-type ATPase.

Things you should learn in graduate school.

Becoming an academic scientist is neither linear nor formulaic. Rather, graduate education is mostly an apprenticeship without a discrete curriculum, clear sign posts of progress, or specific training metrics. In this science and society article I offer a few thoughts from my own experience for what a student beginning this journey might want to learn.

How to enjoy and thrive in graduate school.

Scientific research is an exploration of the unknown. The process is full of uncertainty, missteps, delightful surprises, painful lessons, and ultimately a measure of insight into nature. In this Science and Society article I suggest a few practical strategies that helped me navigate these challenges at the earliest stages of becoming a cell biologist.

EMC rectifies the topology of multipass membrane proteins.

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC's cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome-translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

Control of mRNA fate by its encoded nascent polypeptide.

Cells tightly regulate mRNA processing, localization, and stability to ensure accurate gene expression in diverse cellular states and conditions. Most of these regulatory steps have traditionally been thought to occur before translation by the action of RNA-binding proteins. Several recent discoveries highlight multiple co-translational mechanisms that modulate mRNA translation, localization, processing, and stability. These mechanisms operate by recognition of the nascent protein, which is necessarily coupled to its encoding mRNA during translation. Hence, the distinctive sequence or structure of a particular nascent chain can recruit recognition factors with privileged access to the corresponding mRNA in an otherwise crowded cellular environment. Here, we draw on both well-established and recent examples to provide a conceptual framework for how cells exploit nascent protein recognition to direct mRNA fate. These mechanisms allow cells to dynamically and specifically regulate their transcriptomes in response to changes in cellular states to maintain protein homeostasis.

Mechanism of orphan subunit recognition during assembly quality control.

Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation.

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.

Mechanism of signal-anchor triage during early steps of membrane protein insertion.

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.

The Biogenesis of Multipass Membrane Proteins.

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.

Substrate-driven assembly of a translocon for multipass membrane proteins.

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.

Mechanism of an intramembrane chaperone for multipass membrane proteins.

Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes1,2. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood2. The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel3-9. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism10. Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. Instead, Asterix binds to and redirects the substrate to a location behind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity11. Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endoplasmic reticulum.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

The Function, Structure, and Origins of the ER Membrane Protein Complex.

The endoplasmic reticulum (ER) is the site of membrane protein insertion, folding, and assembly in eukaryotes. Over the past few years, a combination of genetic and biochemical studies have implicated an abundant factor termed the ER membrane protein complex (EMC) in several aspects of membrane protein biogenesis. This large nine-protein complex is built around a deeply conserved core formed by the EMC3-EMC6 subcomplex. EMC3 belongs to the universally conserved Oxa1 superfamily of membrane protein transporters, whereas EMC6 is an ancient, widely conserved obligate partner. EMC has an established role in the insertion of transmembrane domains (TMDs) and less understood roles during the later steps of membrane protein folding and assembly. Several recent structures suggest hypotheses about the mechanism(s) of TMD insertion by EMC, with various biochemical and proteomics studies beginning to reveal the range of EMC's membrane protein substrates.

Mechanism of signal sequence handover from NAC to SRP on ribosomes during ER-protein targeting.

The nascent polypeptide-associated complex (NAC) interacts with newly synthesized proteins at the ribosomal tunnel exit and competes with the signal recognition particle (SRP) to prevent mistargeting of cytosolic and mitochondrial polypeptides to the endoplasmic reticulum (ER). How NAC antagonizes SRP and how this is overcome by ER targeting signals are unknown. Here, we found that NAC uses two domains with opposing effects to control SRP access. The core globular domain prevented SRP from binding to signal-less ribosomes, whereas a flexibly attached domain transiently captured SRP to permit scanning of nascent chains. The emergence of an ER-targeting signal destabilized NAC's globular domain and facilitated SRP access to the nascent chain. These findings elucidate how NAC hands over the signal sequence to SRP and imparts specificity of protein localization.

The mechanisms of integral membrane protein biogenesis.

Roughly one quarter of all genes code for integral membrane proteins that are inserted into the plasma membrane of prokaryotes or the endoplasmic reticulum membrane of eukaryotes. Multiple pathways are used for the targeting and insertion of membrane proteins on the basis of their topological and biophysical characteristics. Multipass membrane proteins span the membrane multiple times and face the additional challenges of intramembrane folding. In many cases, integral membrane proteins require assembly with other proteins to form multi-subunit membrane protein complexes. Recent biochemical and structural analyses have provided considerable clarity regarding the molecular basis of membrane protein targeting and insertion, with tantalizing new insights into the poorly understood processes of multipass membrane protein biogenesis and multi-subunit protein complex assembly.

A unified evolutionary origin for the ubiquitous protein transporters SecY and YidC.

BACKGROUND: Protein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known. RESULTS: The features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY's halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC's sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains. CONCLUSIONS: SecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells.

Identification of a quality-control factor that monitors failures during proteasome assembly.

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.

Translation stress and collided ribosomes are co-activators of cGAS.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to activate the innate immune system. Here, we report the unexpected discovery that cGAS also senses dysfunctional protein production. Purified ribosomes interact directly with cGAS and stimulate its DNA-dependent activity in vitro. Disruption of the ribosome-associated protein quality control (RQC) pathway, which detects and resolves ribosome collision during translation, results in cGAS-dependent ISG expression and causes re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and orthogonal perturbations that result in elevated levels of collided ribosomes and RQC activation cause sub-cellular re-localization of cGAS and ribosome binding in vivo as well. Thus, translation stress potently increases DNA-dependent cGAS activation. These findings have implications for the inflammatory response to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.