Polytopic Membrane Protein Research Articles

Protein insertion into the inner membrane of mitochondria: routes and mechanisms.

The inner membrane of mitochondria contains hundreds of different integral membrane proteins. These proteins transport molecules into and out of the matrix, they carry out multifold catalytic reactions and they promote the biogenesis or degradation of mitochondrial constituents. Most inner membrane proteins are encoded by nuclear genes and synthesized in the cytosol from where they are imported into mitochondria by translocases in the outer and inner membrane. Three different import routes direct proteins into the inner membrane and allow them to acquire their appropriate membrane topology. First, mitochondrial import intermediates can be arrested at the level of the TIM23 inner membrane translocase by a stop-transfer sequence to reach the inner membrane by lateral insertion. Second, proteins can be fully translocated through the TIM23 complex into the matrix from where they insert into the inner membrane in an export-like reaction. Carriers and other polytopic membrane proteins embark on a third insertion pathway: these hydrophobic proteins employ the specialized TIM22 translocase to insert from the intermembrane space (IMS) into the inner membrane. This review article describes these three targeting routes and provides an overview of the machinery that promotes the topogenesis of mitochondrial inner membrane proteins.

A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. Invivo experiments using conditionally depleted E.coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. Invitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.

Characterization of a TatA/TatB binding site on the TatC component of the Escherichia coli twin arginine translocase.

The twin arginine transport (Tat) pathway exports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of chloroplasts. In Escherichia coli and other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organization and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A 'polar' cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6, is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. Characterization of three stably produced TatC variants, P221R, M222R and L225P, each of which is inactive for protein transport, demonstrated that the substitutions did not affect assembly of the Tat receptor. Moreover, the substitutions that we analysed did not abolish TatA or TatB binding to either binding site. Using targeted mutagenesis we introduced bulky substitutions into the TatA binding site. Molecular dynamics simulations and crosslinking analysis indicated that TatA binding at this site was substantially reduced by these amino acid changes, but TatC retained function. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicates that this region of the protein has a critical function.

Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the β barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding

ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 . We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression. Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters. Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Regulation of membrane fluidity by RNF145‐triggered degradation of the lipid hydrolase ADIPOR2

The regulation of membrane lipid composition is critical for cellular homeostasis. Cells are particularly sensitive to phospholipid saturation, with increased saturation causing membrane rigidification and lipotoxicity. How mammalian cells sense membrane lipid composition and reverse fatty acid (FA)‐induced membrane rigidification is poorly understood. Here we systematically identify proteins that differ between mammalian cells fed saturated versus unsaturated FAs. The most differentially expressed proteins were two ER‐resident polytopic membrane proteins: the E3 ubiquitin ligase RNF145 and the lipid hydrolase ADIPOR2. In unsaturated lipid membranes, RNF145 is stable, promoting its lipid‐sensitive interaction, ubiquitination and degradation of ADIPOR2. When membranes become enriched in saturated FAs, RNF145 is rapidly auto‐ubiquitinated and degraded, stabilising ADIPOR2, whose hydrolase activity restores lipid homeostasis and prevents lipotoxicity. We therefore identify RNF145 as a FA‐responsive ubiquitin ligase which, together with ADIPOR2, defines an autoregulatory pathway that controls cellular membrane lipid homeostasis and prevents acute lipotoxic stress.

Cotranslational Biogenesis of Membrane Proteins in Bacteria.

Nascent polypeptides emerging from the ribosome during translation are rapidly scanned and processed by ribosome-associated protein biogenesis factors (RPBs). RPBs cleave the N-terminal formyl and methionine groups, assist cotranslational protein folding, and sort the proteins according to their cellular destination. Ribosomes translating inner-membrane proteins are recognized and targeted to the translocon with the help of the signal recognition particle, SRP, and SRP receptor, FtsY. The growing nascent peptide is then inserted into the phospholipid bilayer at the translocon, an inner-membrane protein complex consisting of SecY, SecE, and SecG. Folding of membrane proteins requires that transmembrane helices (TMs) attain their correct topology, the soluble domains are inserted at the correct (cytoplasmic or periplasmic) side of the membrane, and – for polytopic membrane proteins – the TMs find their interaction partner TMs in the phospholipid bilayer. This review describes the recent progress in understanding how growing nascent peptides are processed and how inner-membrane proteins are targeted to the translocon and find their correct orientation at the membrane, with the focus on biophysical approaches revealing the dynamics of the process. We describe how spontaneous fluctuations of the translocon allow diffusion of TMs into the phospholipid bilayer and argue that the ribosome orchestrates cotranslational targeting not only by providing the binding platform for the RPBs or the translocon, but also by helping the nascent chains to find their correct orientation in the membrane. Finally, we present the auxiliary role of YidC as a chaperone for inner-membrane proteins. We show how biophysical approaches provide new insights into the dynamics of membrane protein biogenesis and raise new questions as to how translation modulates protein folding.

Topology of the Shigella flexneri Enterobacterial Common Antigen polymerase WzyE

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales. In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyESF's periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.

Elevated Levels of an Enzyme Involved in Coenzyme B12 Biosynthesis Kills Escherichia coli.

ABSTRACTCobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called “late” steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5′-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides.

EMC is required for biogenesis of Xport-A, anessential chaperone of Rhodopsin-1 and the TRPchannel.

The ER membrane protein complex (EMC) is required for the biogenesis of a subset of tail anchored (TA) and polytopic membrane proteins, including Rhodopsin-1 (Rh1) and the TRP channel. To understand the physiological implications of EMC-dependent membrane protein biogenesis, we perform a bioinformatic identification of Drosophila TA proteins. From 254 predicted TA proteins, screening in larval eye discs identified two proteins that require EMC for their biogenesis: fan and Xport-A. Fan is required for male fertility in Drosophila and we show that EMC is also required for this process. Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC mutants is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and that EMC-independent Xport-A mutants rescue Rh1 and TRP biogenesis in EMC mutants. Finally, our work also reveals a role for Xport-A in a glycosylation-dependent triage mechanism during Rh1 biogenesis in the endoplasmic reticulum.

Generation of a Polyclonal Antibody against the Mouse Metal Transporter ZIP8.

ZIP8 is a newly identified metal transporter. In human patients, mutations in ZIP8 result in severe manganese deficiency, suggesting a critical role for ZIP8 in regulating systemic manganese homeostasis. In mice, the deletion of ZIP8 recapitulates the symptoms of patients with ZIP8 mutations. However, further studies using mouse models to examine ZIP8′s function were hindered by the lack of suitable antibodies to detect endogenous ZIP8 protein. In this study, we report the design, generation, and validation of a polyclonal antibody against mouse ZIP8. We have demonstrated that the newly generated antibody can be reliably used in immunoblotting analysis to detect endogenous ZIP8 protein in mouse tissues. The successful generation and validation of anti-mouse ZIP8 antibody provide opportunities to further examine the function and regulation of this metal transporter. In addition, our study may provide valuable insights into the future development of antibodies targeting polytopic membrane proteins.

Insights into the Relationship between Cobamide Synthase and the Cell Membrane.

Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The "late steps" of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5' phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis.IMPORTANCESalmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy.

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum’s (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side. The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER membrane has not been identified, despite its importance for N-glycosylation. Building on our ability to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based 'activity correlation profiling' approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients.

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.