Mitochondrial antiviral-signalling protein is a client of the BAG6 protein quality control complex.

The heterotrimeric BAG6 complex coordinates the direct handover of newly synthesised tail-anchored (TA) membrane proteins from an SGTA-bound preloading complex to the endoplasmic reticulum (ER) delivery component TRC40. In contrast, defective precursors, including aberrant TA proteins, form a stable complex with this cytosolic protein quality control factor, enabling such clients to be either productively re-routed or selectively degraded. We identify the mitochondrial antiviral-signalling protein (MAVS) as an endogenous TA client of both SGTA and the BAG6 complex. Our data suggest that the BAG6 complex binds to a cytosolic pool of MAVS before its misinsertion into the ER membrane, from where it can subsequently be removed via ATP13A1-mediated dislocation. This BAG6-associated fraction of MAVS is dynamic and responds to the activation of an innate immune response, suggesting that BAG6 may modulate the pool of MAVS that is available for coordinating the cellular response to viral infection.

His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation.

His domain protein tyrosine phosphatase (HD-PTP; also known as PTPN23) collaborates with endosomal sorting complexes required for transport (ESCRTs) to sort endosomal cargo into intralumenal vesicles, forming the multivesicular body (MVB). Completion of MVB sorting is accompanied by maturation of the endosome into a late endosome, an event that requires inactivation of the early endosomal GTPase Rab5 (herein referring to generically to all isoforms). Here, we show that HD-PTP links ESCRT function with endosomal maturation. HD-PTP depletion prevents MVB sorting, while also blocking cargo from exiting Rab5-rich endosomes. HD-PTP-depleted cells contain hyperphosphorylated Rabaptin-5 (also known as RABEP1), a cofactor for the Rab5 guanine nucleotide exchange factor Rabex-5 (also known as RABGEF1), although HD-PTP is unlikely to directly dephosphorylate Rabaptin-5. In addition, HD-PTP-depleted cells exhibit Rabaptin-5-dependent hyperactivation of Rab5. HD-PTP binds directly to Rabaptin-5, between its Rabex-5- and Rab5-binding domains. This binding reaction involves the ESCRT-0/ESCRT-III binding site in HD-PTP, which is competed for by an ESCRT-III peptide. Jointly, these findings indicate that HD-PTP may alternatively scaffold ESCRTs and modulate Rabex-5-Rabaptin-5 activity, thereby helping to coordinate the completion of MVB sorting with endosomal maturation.

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis.

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2, the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation and/or insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum antiviral agents.This article has an associated First Person interview with the first author of the paper.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2 the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation/insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum, antiviral agents.

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control.

BACKGROUND: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. RESULTS: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. CONCLUSION: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region.

Loss of the golgin GM130 causes Golgi disruption, Purkinje neuron loss, and ataxia in mice.

The Golgi apparatus lies at the heart of the secretory pathway where it is required for secretory trafficking and cargo modification. Disruption of Golgi architecture and function has been widely observed in neurodegenerative disease, but whether Golgi dysfunction is causal with regard to the neurodegenerative process, or is simply a manifestation of neuronal death, remains unclear. Here we report that targeted loss of the golgin GM130 leads to a profound neurological phenotype in mice. Global KO of mouse GM130 results in developmental delay, severe ataxia, and postnatal death. We further show that selective deletion of GM130 in neurons causes fragmentation and defective positioning of the Golgi apparatus, impaired secretory trafficking, and dendritic atrophy in Purkinje cells. These cellular defects manifest as reduced cerebellar size and Purkinje cell number, leading to ataxia. Purkinje cell loss and ataxia first appear during postnatal development but progressively worsen with age. Our data therefore indicate that targeted disruption of the mammalian Golgi apparatus and secretory traffic results in neuronal degeneration in vivo, supporting the view that Golgi dysfunction can play a causative role in neurodegeneration.

The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway.

Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER-Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2).

Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210.

Golgins are extended coiled-coil proteins believed to participate in membrane-tethering events at the Golgi apparatus. However, the importance of golgin-mediated tethering remains poorly defined, and alternative functions for golgins have been proposed. Moreover, although golgins bind to Rab GTPases, the functional significance of Rab binding has yet to be determined. In this study, we show that depletion of the golgin GMAP-210 causes a loss of Golgi cisternae and accumulation of numerous vesicles. GMAP-210 function in vivo is dependent upon its ability to tether membranes, which is mediated exclusively by the amino-terminal ALPS motif. Binding to Rab2 is also important for GMAP-210 function, although it is dispensable for tethering per se. GMAP-210 length is also functionally important in vivo. Together our results indicate a key role for GMAP-210-mediated membrane tethering in maintaining Golgi structure and support a role for Rab2 binding in linking tethering with downstream docking and fusion events at the Golgi apparatus.

Elevation of proteasomal substrate levels sensitizes cells to apoptosis induced by inhibition of proteasomal deubiquitinases.

Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s) underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system.

Biochemical analysis of secretory trafficking in mammalian cells.

Protein trafficking within the secretory pathway of mammalian cells is amenable to analysis by biochemical methods. This can be achieved by monitoring posttranslational modifications that occur naturally within the secretory pathway, or by measuring the delivery of cargo to the cell surface or extracellular medium. These approaches can be combined with additional manipulations such as specific temperature blocks that permit analysis of distinct trafficking steps. Biochemical analysis is advantageous in that it permits both a sensitive and quantitative measure of trafficking along the pathway. The methods discussed in this chapter permit the analysis of trafficking of both endogenous cargo proteins and ectopically expressed model cargos, which can be followed using either Western blotting or metabolic pulse-chase approaches. These methods are relatively straightforward and suitable for use in most modern cell biology laboratories. In addition to the well-established methods that we describe here in detail, we also refer to the development of more recent tailored approaches that add further to the arsenal of tools that can be used to assess trafficking in the secretory pathway.

OST4 is a subunit of the mammalian oligosaccharyltransferase required for efficient N-glycosylation.

The eukaryotic oligosaccharyltransferase (OST) is a membrane-embedded protein complex that catalyses the N-glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum (ER), a highly conserved biosynthetic process that enriches protein structure and function. All OSTs contain a homologue of the catalytic STT3 subunit, although in many cases this is assembled with several additional components that influence function. In S. cerevisiae, one such component is Ost4p, an extremely small membrane protein that appears to stabilise interactions between subunits of assembled OST complexes. OST4 has been identified as a putative human homologue, but to date neither its relationship to the OST complex, nor its role in protein N-glycosylation, have been directly addressed. Here, we establish that OST4 is assembled into native OST complexes containing either the catalytic STT3A or STT3B isoforms. Co-immunoprecipitation studies suggest that OST4 associates with both STT3 isoforms and with ribophorin I, an accessory subunit of mammalian OSTs. These presumptive interactions are perturbed by a single amino acid change in the transmembrane region of OST4. Using siRNA knockdowns and native gel analysis, we show that OST4 plays an important role in maintaining the stability of native OST complexes. Hence, upon OST4 depletion well-defined OST complexes are partially destabilised and a novel ribophorin I-containing subcomplex can be detected. Strikingly, cells depleted of either OST4 or STT3A show a remarkably similar defect in the N-glycosylation of endogenous prosaposin. We conclude that OST4 most likely promotes co-translational N-glycosylation by stabilising STT3A-containing OST isoforms.

Positional editing of transmembrane domains during ion channel assembly.

The integration of transmembrane (TM)-spanning regions of many channels and ion transporters is potentially compromised by the presence of polar and charged residues required for biological function. Although the two TMs of the ATP-gated ion channel subunit P2X2 each contain charged/polar amino acids, we found that each TM is efficiently membrane inserted when it is analysed in isolation, and uncovered no evidence for cooperativity between these two TMs during P2X2 integration. However, using minimal N-glycosylation distance mapping, we find that the positioning of TM2 in newly synthesized P2X2 monomers is distinct from that seen in subunits of the high-resolution structures of assembled homologous trimers. We conclude that P2X2 monomers are initially synthesised at the endoplasmic reticulum in a distinct conformation, where the extent of the TM-spanning regions is primarily defined by the thermodynamic cost of their membrane integration at the Sec61 translocon. In this model, TM2 of P2X2 subsequently undergoes a process of positional editing within the membrane that correlates with trimerisation of the monomer, a process requiring specific polar/charged residues in both TM1 and TM2. We postulate that the assembly process offsets any energetic cost of relocating TM2, and find evidence that positional editing of TM2 in the acid-sensing ion channel (ASIC1a) is even more pronounced than that observed for P2X2. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains. We propose that the orchestrated repositioning of TM segments during subunit oligomerisation plays an important role in generating the functional architecture of active ion channels, and suggest that the regulation of this underappreciated biosynthetic step may provide an elegant mechanism for maintaining ER homeostasis.

The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation.

Protein N-glycosylation is an essential modification that occurs in all eukaryotes and is catalysed by the oligosaccharyltransferase (OST) in the endoplasmic reticulum. Comparative studies have clearly shown that eukaryotic STT3 proteins alone can fulfil the enzymatic requirements for N-glycosylation, yet in many cases STT3 homologues form stable complexes with a variety of non-catalytic OST subunits. Whereas some of these additional components might play a structural role, others appear to increase or modulate N-glycosylation efficiency for certain precursors. Here, we have analysed the roles of three non-catalytic mammalian OST components by studying the consequences of subunit-specific knockdowns on the stability and enzymatic activity of the OST complex. Our results demonstrate that OST48 and DAD1 are required for the assembly of both STT3A- and STT3B-containing OST complexes. The structural perturbations of these complexes we observe in OST48- and DAD1-depleted cells underlie their pronounced hypoglycosylation phenotypes. Thus, OST48 and DAD1 are global modulators of OST stability and hence N-glycosylation. We show that KCP2 also influences protein N-glycosylation, yet in this case, the effect of its depletion is substrate specific, and is characterised by the accumulation of a novel STT3A-containing OST subcomplex. Our results suggest that KCP2 acts to selectively enhance the OST-dependent processing of specific protein precursors, most likely co-translational substrates of STT3A-containing complexes, highlighting the potential for increased complexity of OST subunit composition in higher eukaryotes.

Keratinocyte-associated protein 2 is a bona fide subunit of the mammalian oligosaccharyltransferase.

The oligosaccharyltransferase (OST) complex catalyses the N-glycosylation of polypeptides entering the endoplasmic reticulum, a process essential for the productive folding and trafficking of many secretory and membrane proteins. In eukaryotes, the OST typically comprises a homologous catalytic STT3 subunit complexed with several additional components that are usually conserved, and that often function to modulate N-glycosylation efficiency. By these criteria, the status of keratinocyte-associated protein 2 (KCP2) was unclear: it was found to co-purify with the canine OST suggesting it is part of the complex but, unlike most other subunits, no potential homologues are apparent in Saccharomyces cerevisiae. In this study we have characterised human KCP2 and show that the predominant species results from an alternative initiation of translation to form an integral membrane protein with three transmembrane spans. KCP2 localises to the endoplasmic reticulum, consistent with a role in protein biosynthesis, and has a functional KKxx retrieval signal at its cytosolic C-terminus. Native gel analysis suggests that the majority of KCP2 assembles into a distinct ~500 kDa complex that also contains several bona fide OST subunits, most notably the catalytic STT3A isoform. Co-immunoprecipitation studies confirmed a robust and specific physical interaction between KCP2 and STT3A, and revealed weaker associations with both STT3B and OST48. Taken together, these data strongly support the proposal that KCP2 is a newly identified subunit of the N-glycosylation machinery present in a subset of eukaryotes.

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response.

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ES(I) (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ES(I) causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.

Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum.

Production and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-associated degradation (ERAD) eeyarestatin 1 (ES(I)) is a potent inhibitor of protein translocation. We have characterised this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ES(I) targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ES(I) acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ES(I), and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.

Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein.

Missense mutations in human PLP1, the gene encoding myelin proteolipid protein (PLP), cause dysmyelinating Pelizaeus-Merzbacher disease of varying severity. Although disease pathology has been linked to retention of misfolded PLP in the endoplasmic reticulum (ER) and induction of the unfolded protein response (UPR), the molecular mechanisms that govern phenotypic heterogeneity remain poorly understood. To address this issue, we examined the cellular response to missense mutants of PLP that are associated with distinct disease phenotypes. We found that the mild-disease-associated mutants, W162L and G245A, were cleared from the ER comparatively quickly via proteasomal degradation and/or ER exit. By contrast, the more ;aggressive' A242V mutant, which causes severe disease, was significantly more stable, accumulated at the ER and resulted in a specific activation of the UPR. On the basis of these findings, we propose that the rate at which mutant PLP proteins are cleared from the ER modulates disease severity by determining the extent to which the UPR is activated.