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Coronaviruses are characterized by their progeny assembly and budding in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). Our previous studies demonstrated that truncation of 9 amino acids in the cytoplasmic tail (CT) of the infectious bronchitis virus (IBV) spike (S) protein impairs its localization to the ERGIC, resulting in increased expression at the plasma membrane. However, the precise mechanism underlying this phenomenon remained elusive. In this study, we provide evidence that the IBV S protein could utilize coatomer protein-I (COPI)-coated vesicles for retrograde transport from the Golgi to the endoplasmic reticulum (ER). We identified the KKSV motif as the critical binding site within the CT domain of IBV S protein for COPI interaction. Further analysis reveals that IBV infection does not modulate host COPI expression. However, when COPI expression is disrupted, a higher proportion of S protein escapes to the plasma membrane. Moreover, inhibition of COPI-mediated transport during viral infection severely impairs progeny virion production and leads to increased S protein accumulation at the plasma membrane, inducing cell-cell fusion and syncytia formation. Our findings contribute to a deeper understanding of S protein intracellular trafficking during coronavirus infection, and offer valuable insights into the molecular mechanisms of viral replication and host cell biology.IMPORTANCEViruses hijack or modify host cellular machiner y and associated pathways to facilitate their own replication. Here, we demonstrate that the infectious bronchitis virus (IBV) S protein directly interacts with coatomer protein-I (COPI)-coated vesicles through the KKSV motif in its cytoplasmic tail. COPI-coated vesicles mediate the retrograde transport of S protein from the Golgi apparatus to the endoplasmic reticulum-Golgi intermediate compartment, where viral particle assembly occurs. Our findings not only advance our understanding of IBV S protein trafficking mechanisms but also provide valuable insights for developing more effective vaccine strategies.

SARS-CoV-2 undergoes budding within the lumen of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and the progeny virions are delivered to the cell surface via vesicular transport. However, the molecular mechanisms remain poorly understood. Using three-dimensional electron microscopic analysis, such as array tomography and electron tomography, we found that virion-transporting vesicles possessed protein coats on their membrane and demonstrated that the protein coat was coatomer complex I (COPI). During the later stages of SARS-CoV-2 infection, we observed a notable alteration in the distribution of COPI and ERGIC throughout the cytoplasm, suggesting their potential involvement in virus replication. Depletion of COPB2, a key component of COPI, led to the confinement of SARS-CoV-2 progeny virions within the ERGIC at the perinuclear region. While the expression levels of viral proteins within cells were comparable, this depletion significantly reduced the efficiency of virion release, leading to the significant reduction of viral replication. Hence, our findings suggest COPI as a critical player in facilitating the transport of SARS-CoV-2 progeny virions from the ERGIC. Thus, COPI could be a promising target for the development of antivirals against SARS-CoV-2., Importance: SARS-CoV-2 virions are synthesized within the ERGIC and are transported to the cell surface via vesicular transport for release. However, the precise mechanisms remain unclear. Through various electron microscopic techniques, we identified the presence of COPI on virion-transporting vesicles. Alterations in the distribution of COPI and ERGIC in SARS-CoV-2 infected cells are evident, suggesting their involvement in virus replication. When COPB2, a component of COPI, is depleted, progeny virions become trapped within the ERGIC, leading to a reduction in the efficiency of virion release. These findings highlight COPI's crucial role in mediating SARS-CoV-2 vesicular transport from the ERGIC and suggest it as a potential antiviral target., Competing Interests: The authors declare no conflict of interest.

Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. Here, we used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Stimulator of Interferon Genes (STING) serves as a pivotal mediator in the innate immune signaling pathway, transducing signals from various DNA receptors and playing a crucial role in natural immune processes. During cellular quiescence, STING protein resides in the endoplasmic reticulum (ER), and its activation typically occurs through the cGAS-STING signaling pathway. Upon activation, STING protein is transported to the Golgi apparatus, thereby initiating downstream signaling cascades. Vesicular transport serves as the primary mechanism for STING protein trafficking between the ER and Golgi apparatus, with COPII mediating anterograde transport from the ER to Golgi apparatus, while COPI is responsible for retrograde transport. Numerous factors influence these transport processes, thereby exerting either promoting or inhibitory effects on STING protein expression. Upon reaching the Golgi apparatus, to prevent over-activation, STING protein is transported to post-Golgi compartments for degradation. In addition to the conventional lysosomal degradation pathway, ESCRT has also been identified as one of the degradation pathways for STING protein. This review summarizes the recent findings on the membrane trafficking pathways of STING, highlighting their contributions to the regulation of cytokine production, the activation of immune cells, and the coordination of immune signaling pathways., 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 © 2024 Elsevier Ltd. All rights reserved.)

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV., Importance: Arenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member lymphocytic choriomeningitis virus (LCMV) being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. A detailed understanding of how arenaviruses subvert host cell processes to complete their multiplication cycle is incomplete. Here, using a combination of gene ablation and pharmacological inhibition techniques, we showed that host cellular COPI and AP-4 complexes, with native roles in cellular vesicular transport, were required for efficient LCMV growth. We further showed these complexes acted on late stages of the multiplication cycle, post-gene expression, with a significant impact on infectious virus egress. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal critical cellular trafficking pathways required during infection., Competing Interests: The authors declare no conflict of interest.

The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Importance: The Golgi is an essential eukaryotic organelle and a major place for protein sorting and glycosylation. Among apicomplexan parasites, Toxoplasma gondii retains the most developed Golgi structure and produces many glycosylated factors necessary for parasite survival. Despite its importance, Golgi function received little attention in the past. In the current study, we identified and characterized the conserved oligomeric Golgi complex and its novel partners critical for protein transport in T. gondii tachyzoites. Our results suggest that T. gondii broadened the role of the conserved elements and reinvented the missing components of the trafficking machinery to accommodate the specific needs of the opportunistic parasite T. gondii ., Competing Interests: The authors declare no conflict of interest.

Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst. The Golgi-dependent secretory pathway is thought to be responsible for the trafficking of all major membrane proteins. However, our recent findings in Aspergillus nidulans showed that several plasma membrane cargoes, such as transporters and receptors, follow a sorting route that seems to bypass Golgi functioning. To gain insight on membrane trafficking and specifically Golgi-bypass, here we used proximity dependent biotinylation (PDB) coupled with data-independent acquisition mass spectrometry (DIA-MS) for identifying transient interactors of the UapA transporter. Our assays, which included proteomes of wild-type and mutant strains affecting ER-exit or endocytosis, identified both expected and novel interactions that might be physiologically relevant to UapA trafficking. Among those, we validated, using reverse genetics and fluorescence microscopy, that COPI coatomer is essential for ER-exit and anterograde trafficking of UapA and other membrane cargoes. We also showed that ArfA Arf1 GTPase activating protein (GAP) Glo3 contributes to UapA trafficking at increased temperature. This is the first report addressing the identification of transient interactions during membrane cargo biogenesis using PDB and proteomics coupled with fungal genetics. Our work provides a basis for dissecting dynamic membrane cargo trafficking via PDB assays., 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 Inc. All rights reserved.)

Coat protein complex I (COPI) is best known for its role in Golgi-endoplasmic reticulum (ER) trafficking, responsible for the retrograde transport of ER-resident proteins. The ER is crucial to neuronal function, regulating Ca 2+ homeostasis and the distribution and function of other organelles such as endosomes, peroxisomes, and mitochondria via functional contact sites. Here we demonstrate that disruption of COPI results in mitochondrial dysfunction in Drosophila axons and human cells. The ER network is also disrupted, and the neurons undergo rapid degeneration. We demonstrate that mitochondria-ER contact sites (MERCS) are decreased in COPI-deficient axons, leading to Ca 2+ dysregulation, heightened mitophagy, and a decrease in respiratory capacity. Reintroducing MERCS is sufficient to rescue not only mitochondrial distribution and Ca 2+ uptake but also ER morphology, dramatically delaying neurodegeneration. This work demonstrates an important role for COPI-mediated trafficking in MERC formation, which is an essential process for maintaining axonal integrity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Transport models are extremely important to map thousands of proteins and their interactions inside a cell. The transport pathways of luminal and at least initially soluble secretory proteins synthesized in the endoplasmic reticulum can be divided into two groups: the so-called constitutive secretory pathway and regulated secretion (RS) pathway, in which the RS proteins pass through the Golgi complex and are accumulated into storage/secretion granules (SGs). Their contents are released when stimuli trigger the fusion of SGs with the plasma membrane (PM). In specialized exocrine, endocrine, and nerve cells, the RS proteins pass through the baso-lateral plasmalemma. In polarized cells, the RS proteins secrete through the apical PM. This exocytosis of the RS proteins increases in response to external stimuli. Here, we analyze RS in goblet cells to try to understand the transport model that can be used for the explanation of the literature data related to the intracellular transport of their mucins.

The eukaryotic phylum Parabasalia is composed primarily of anaerobic, endobiotic organisms such as the veterinary parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis, the latter causing the most prevalent, non-viral, sexually transmitted disease world-wide. Although a parasitic lifestyle is generally associated with a reduction in cell biology, T. vaginalis provides a striking counter-example. The 2007 T. vaginalis genome paper reported a massive and selective expansion of encoded proteins involved in vesicle trafficking, particularly those implicated in the late secretory and endocytic systems. Chief amongst these were the hetero-tetrameric adaptor proteins or 'adaptins', with T. vaginalis encoding ∼3.5 times more such proteins than do humans. The provenance of such a complement, and how it relates to the transition from a free-living or endobiotic state to parasitism, remains unclear. In this study, we performed a comprehensive bioinformatic and molecular evolutionary investigation of the heterotetrameric cargo adaptor-derived coats, comparing the molecular complement and evolution of these proteins between T. vaginalis, T. foetus and the available diversity of endobiotic parabasalids. Notably, with the recent discovery of Anaeramoeba spp. as the free-living sister lineage to all parabasalids, we were able to delve back to time points earlier in the lineage's history than ever before. We found that, although T. vaginalis still encodes the most HTAC subunits amongst parabasalids, the duplications giving rise to the complement took place more deeply and at various stages across the lineage. While some duplications appear to have convergently shaped the parasitic lineages, the largest jump is in the transition from free-living to endobiotic lifestyle with both gains and losses shaping the encoded complement. This work details the evolution of a cellular system across an important lineage of parasites and provides insight into the evolutionary dynamics of an example of expansion of protein machinery, counter to the more common trends observed in many parasitic systems., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Graphitic carbon nitride (gCN) is a promising n -type semiconductor widely investigated for photo-assisted water splitting, but less studied for the (photo)electrochemical degradation of aqueous organic pollutants. In these fields, attractive perspectives for advancements are offered by a proper engineering of the material properties, e.g., by depositing gCN onto conductive and porous scaffolds, tailoring its nanoscale morphology, and functionalizing it with suitable cocatalysts. The present study reports on a simple and easily controllable synthesis of gCN flakes on Ni foam substrates by electrophoretic deposition (EPD), and on their eventual decoration with Co-based cocatalysts [CoO, CoFe 2 O 4 , cobalt phosphate (CoPi)] via radio frequency (RF)-sputtering or electrodeposition. After examining the influence of processing conditions on the material characteristics, the developed systems are comparatively investigated as (photo)anodes for water splitting and photoelectrocatalysts for the degradation of a recalcitrant water pollutant [potassium hydrogen phthalate (KHP)]. The obtained results highlight that while gCN decoration with Co-based cocatalysts boosts water splitting performances, bare gCN as such is more efficient in KHP abatement, due to the occurrence of a different reaction mechanism. The related insights, provided by a multi-technique characterization, may provide valuable guidelines for the implementation of active nanomaterials in environmental remediation and sustainable solar-to-chemical energy conversion.

COPI-coated vesicles mediate transport between Golgi stacks and retrograde transport from the Golgi to the endoplasmic reticulum. The COPI coat exists as a stable heptameric complex in the cytosol termed coatomer and is recruited en bloc to the membrane for vesicle formation. Recruitment of COPI onto membranes is mediated by the Arf family of small GTPases, which, in their GTP-bound state, bind both membrane and coatomer. Arf GTPases also influence cargo selection, vesicle scission and vesicle uncoating. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) regulate nucleotide binding by Arf GTPases. To understand the mechanism of COPI-coated vesicle trafficking, it is necessary to characterize the interplay between coatomer and Arf GTPases and their effectors. It is also necessary to understand interactions between coatomer and cargo, cargo adaptors/receptors and tethers facilitating binding to the target membrane. Here, we summarize current knowledge of COPI coat protein structure; we describe how structural and biochemical studies contributed to this knowledge; we review mechanistic insights into COPI vesicle biogenesis and disassembly; and we discuss the potential to answer open questions in the field., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A common approach for the photoelectrochemical (PEC) splitting of water relies on the application of WO 3 porous electrodes sensitized with BiVO 4 acting as a visible photoanode semiconductor. In this work, we propose a new architecture of photoelectrodes consisting of supported multishell nanotubes (NTs) fabricated by a soft-template approach. These NTs are formed by a concentric layered structure of indium tin oxide (ITO), WO 3 , and BiVO 4 , together with a final thin layer of cobalt phosphate (CoPi) co-catalyst. The photoelectrode manufacturing procedure is easily implementable at a large scale and successively combines the thermal evaporation of single crystalline organic nanowires (ONWs), the magnetron sputtering deposition of ITO and WO 3 , and the solution dripping and electrochemical deposition of, respectively, BiVO 4 and CoPi, plus the annealing in air under mild conditions. The obtained NT electrodes depict a large electrochemically active surface and outperform the efficiency of equivalent planar-layered electrodes by more than one order of magnitude. A thorough electrochemical analysis of the electrodes illuminated with blue and solar lights demonstrates that the characteristics of the WO 3 /BiVO 4 Schottky barrier heterojunction control the NT electrode efficiency, which depended on the BiVO 4 outer layer thickness and the incorporation of the CoPi electrocatalyst. These results support the high potential of the proposed soft-template methodology for the large-area fabrication of highly efficient multishell ITO/WO 3 /BiVO 4 /CoPi NT electrodes for the PEC splitting of water.

The Coat Protein I (COPI) complex forms vesicles from Golgi membrane for retrograde transport among the Golgi stacks, and also from the Golgi to the endoplasmic reticulum (ER). We have been elucidating the mechanistic details of COPI vesicle formation through a reconstitution system that involves the incubation of Golgi membrane with purified components. This approach has enabled us recently to gain new insight into how certain lipids are critical for the fission stage of COPI vesicle formation. Lipid geometry has been proposed to act in the formation of transport carriers by promoting membrane curvature. However, evidence for this role has come from studies using simplified membranes, while confirmation in the more physiologic setting of native membranes has been challenging, as such membranes contain a complex composition of lipids and proteins. We have recently refined the COPI reconstitution system to overcome this experimental obstacle. This has led us to identify an unanticipated type of lipid geometry needed for COPI vesicle fission. This chapter describes the approach that we have developed to enable this discovery. The methodologies include: (i) preparation Golgi membrane from cells that are deficient in a particular lipid enzyme activity and (ii) functional rescue of this deficiency by introducing the product of the lipid enzyme, with experiments being performed at the in vitro level to gain mechanistic clarity and at the in vivo level to confirm physiologic relevance., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of β-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Golgi-associated retrograde protein (GARP) is an evolutionary conserved heterotetrameric protein complex that tethers endosome-derived vesicles and is vital for Golgi glycosylation. Microscopy and proteomic approaches were employed to investigate defects in Golgi physiology in RPE1 cells depleted for the GARP complex. Both cis and trans -Golgi compartments were significantly enlarged in GARP-knock-out (KO) cells. Proteomic analysis of Golgi-enriched membranes revealed significant depletion of a subset of Golgi residents, including Ca 2+ binding proteins, enzymes, and SNAREs. Validation of proteomics studies revealed that SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, were significantly depleted in GARP-KO cells. Finding that GARP-KO is more deleterious to Golgi physiology than deletion of GARP-sensitive v-SNAREs, prompted a detailed investigation of COPI trafficking machinery. We discovered that in GARP-KO cells COPI is significantly displaced from the Golgi and partially relocalized to the ER-Golgi intermediate compartment (ERGIC). Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1, and BIG1 are also relocated to off-Golgi compartments. We propose that the dysregulation of COPI machinery, along with the depletion of Golgi v-SNAREs and alteration of Golgi Ca 2+ homeostasis, are the major driving factors for the depletion of Golgi resident proteins, structural alterations, and glycosylation defects in GARP deficient cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khakurel, Kudlyk, Pokrovskaya, D’Souza and Lupashin.)

The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry through associating with ACE2 on host cells. Intracellular trafficking and palmitoylation of S protein are required for its function. The short cytoplasmic tail of S protein plays a key role in the intracellular trafficking, which contains the binding site for the host trafficking proteins such as COPI, COPII and SNX27. This cytoplasmic tail also contains the palmitoylation sites of S protein. Protein palmitoylation modification of S protein could be catalyzed by a family of zinc finger DHHC domain-containing protein palmitoyltransferases (ZDHHCs). The intracellular trafficking and membrane location facilitate surface expression of S protein and assembly of progeny virions. In this review, we summarize the function of S protein cytoplasmic tail in transportation and localization. S protein relies on intracellular trafficking pathways and palmitoylation modification to facilitate the life cycle of SARS-CoV-2, meanwhile it could interfere with the host transport pathways. The interplay between S protein and intracellular trafficking proteins could partially explain the acute symptoms or Long-COVID complications in multiple organs of COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Liu and Zhang.)

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae . The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of β'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization., Competing Interests: SD, PX, NH, ND, JB, BX, JD, ES, LJ, JM, TG No competing interests declared, (© 2022, Date et al.)

Plant Golgi apparatus serves as the central station of the secretory pathway and is the site where protein modification and cell wall matrix polysaccharides synthesis occur. The polarized and stacked cisternal structure is a prerequisite for Golgi function. Our understanding of Golgi structure maintenance and trafficking are largely obtained from mammals and yeast, yet, plant Golgi has many different aspects. In this review, we summarize the key players in Golgi maintenance demonstrated by genetic studies in plants, which function in ER-Golgi, intra-Golgi and post-Golgi transport pathways. Among these, we emphasize on players in intra-Golgi trafficking., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rui, Tan, Liu and Bao.)

Purpose: This study aimed to describe the phenotypic and molecular characteristics of ARCN1-related syndrome., Methods: Patients with ARCN1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient., Results: In total, we identified 14 cases of ARCN1-related syndrome, (9 pediatrics, and 5 fetal cases from 3 families). The clinical features these newly identified cases were compared to 6 previously reported cases for a total of 20 cases. Intrauterine growth restriction, micrognathia, and short stature were present in all patients. Other common features included prematurity (11/15, 73.3%), developmental delay (10/14, 71.4%), genitourinary malformations in males (6/8, 75%), and microcephaly (12/15, 80%). Novel features of ARCN1-related syndrome included transient liver dysfunction and specific glycosylation abnormalities during illness, giant cell hepatitis, hepatoblastoma, cataracts, and lethal skeletal manifestations. Developmental delay was seen in 73% of patients, but only 3 patients had intellectual disability, which is less common than previously reported., Conclusion: ARCN1-related syndrome presents with a wide clinical spectrum ranging from a severe embryonic lethal syndrome to a mild syndrome with intrauterine growth restriction, micrognathia, and short stature without intellectual disability. Patients with ARCN1-related syndrome should be monitored for liver dysfunction during illness, cataracts, and hepatoblastoma. Additional research to further define the phenotypic spectrum and possible genotype-phenotype correlations are required., Competing Interests: Conflict of Interest The following authors declare no conflicts of interest: A.L.R., J.G., Hi.H., A.M.A., S.H., C.S., S.C., E.B., D.L., Y.K., A.B., S.E.C., A.P., J.O., M.K.K., K.N., K.K., T.O., T.B., P.R., B.K., J.R.M., J.A.S., V.S., N.S., Ha.H., K.S., R.T., W.R.W., K.V.W., K.T., K.C., A.E., J.L., T.M., M.H., A.C.E., and K.I. J.W. receives research support from Gilead Sciences, Inc; AbbVie; and Alexion and has consulting relationship with Gilead Sciences, Inc. E.T., R.W., and Y.S. are employees of GeneDx, Inc., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

The Golgi complex is the central station of the secretory pathway. Knowledge about the mechanisms of intra-Golgi transport is inconsistent. Here, we compared the explanatory power of the cisterna maturation-progression model and the kiss-and-run model. During intra-Golgi transport, conventional cargoes undergo concentration and form cisternal distensions or distinct membrane domains that contain only one membrane cargo. These domains and distension are separated from the rest of the Golgi cisternae by rows of pores. After the arrival of any membrane cargo or a large cargo aggregate at the Golgi complex, the cis -Golgi SNAREs become enriched within the membrane of cargo-containing domains and then replaced by the trans-Golgi SNAREs. During the passage of these domains, the number of cisternal pores decreases. Restoration of the cisternal pores is COPI-dependent. Our observations are more in line with the kiss-and-run model.

Protein glycosylation in the Golgi is a sequential process that requires proper distribution of transmembrane glycosyltransferase enzymes in the appropriate Golgi compartments. Some of the cytosolic machinery required for the steady-state localization of some Golgi enzymes are known but existing models do not explain how many of these enzymes are localized. Here, we uncover the role of an integral membrane protein in yeast, Erd1, as a key facilitator of Golgi glycosyltransferase recycling by directly interacting with both the Golgi enzymes and the cytosolic receptor, Vps74. Loss of Erd1 function results in mislocalization of Golgi enzymes to the vacuole/lysosome. We present evidence that Erd1 forms an integral part of the recycling machinery and ensures productive recycling of several early Golgi enzymes. Our work provides new insights on how the localization of Golgi glycosyltransferases is spatially and temporally regulated, and is finely tuned to the cues of Golgi maturation., Competing Interests: RS, CH, BS, CC, JF, SE No competing interests declared, (© 2021, Sardana et al.)

Conversion between phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate on endosomal membranes is critical for the maturation of early endosomes to late endosomes/lysosomes and is regulated by the PIKfyve-Vac14-Fig4 complex. Despite the importance of this complex for endosomal homeostasis and vesicular trafficking, there is little known about how its activity is regulated or how it interacts with other cellular proteins. Here, we screened for the cellular interactome of Vac14 and Fig4 using proximity-dependent biotin labeling (BioID). After independently screening the interactomes of Vac14 and Fig4, we identified 89 high-confidence protein hits shared by both proteins. Network analysis of these hits revealed pathways with known involvement of the PIKfyve-Vac14-Fig4 complex, including vesicular organization and PI3K/Akt signaling, as well as novel pathways including cell cycle and mitochondrial regulation. We also identified subunits of coatomer complex I (COPI), a Golgi-associated complex with an emerging role in endosomal dynamics. Using proximity ligation assays, we validated the interaction between Vac14 and COPI subunit COPB1 and between Vac14 and Arf1, a GTPase required for COPI assembly. In summary, this study used BioID to comprehensively map the Vac14-Fig4 interactome, revealing potential roles for these proteins in diverse cellular processes and pathways, including preliminary evidence of an interaction between Vac14 and COPI. Data are available via ProteomeXchange with the identifier PXD027917.

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits and demonstrate an accumulation of 'coat-less' vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation., Competing Interests: TQ, JW, EH, NP, WL, PK, JW, LM, MK, JM, EL, GP, PM No competing interests declared, (© 2021, Quidwai et al.)

Background: Cisd1 and Cisd2 proteins share very similar structures with an N-terminal membrane-anchoring domain and a C-terminal cytosolic domain containing an iron-cluster binding domain and ending with a C-terminal KKxx sequence. Despite sharing a similar structure, Cisd1 and Cisd2 are anchored to different compartments: mitochondria for Cisd1 and endoplasmic reticulum for Cisd2. The aim of this study was to identify the protein motifs targeting Cisd2 to the ER and ensuring its retention in this compartment., Results: We used new recombinant antibodies to localize Cisd1 and Cisd2 proteins, as well as various protein chimeras. Cisd2 is targeted to the ER by its N-terminal sequence. It is then retained in the ER by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif, and of an ER-targeting transmembrane domain. As previously reported for Cisd1, Cisd2 can alter the morphology of the compartment in which it accumulates., Conclusion: Although they share a very similar structure, Cisd1 and Cisd2 use largely different intracellular targeting motifs to reach their target compartment (mitochondria and endoplasmic reticulum, respectively)., (© 2021. The Author(s).)

Protein coats, important for vesicular trafficking in eukaryotic cells, help shape membranes and package cargo. But their dynamic construction cannot be fully understood until the distinct steps of their assembly in their native intracellular context at molecular resolution can be visualized. For this, correlative light and electron microscopy (CLEM) is an essential tool. Here, we discuss how emerging CLEM techniques have been used to study the assembly of protein coats inside cells. We review how current and developing CLEM technologies are poised to answer fundamental questions of protein coat architecture at the nanoscale., Competing Interests: Conflict of interest statement Nothing declared., (Published by Elsevier Ltd.)

Molecular switches of the ADP-ribosylation factor (ARF) GTPase family coordinate intracellular trafficking at all sorting stations along the secretory pathway, from the ER-Golgi-intermediate compartment (ERGIC) to the plasma membrane (PM). Their GDP-GTP switch is essential to trigger numerous processes, including membrane deformation, cargo sorting and recruitment of downstream coat proteins and effectors, such as lipid modifying enzymes. While ARFs (in particular ARF1) had mainly been studied in the context of coat protein recruitment at the Golgi, COPI/clathrin-independent roles have emerged in the last decade. Here we review the roles of human ARF1-5 GTPases in cellular trafficking with a particular emphasis on their roles in post-Golgi secretory trafficking and in sorting in the endo-lysosomal system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Adarska, Wong-Dilworth and Bottanelli.)

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement., Competing Interests: Declaration of interests C.S.X. and H.F.H. have a U.S. patent 10,600,615 of the enhanced FIB-SEM system used in this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. Here, we reveal a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, our in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: The advent of Cone Beam Computed Tomography (CBCT) in endodontics has enhanced the diagnosis of periapical radiolucencies and the assessment of endodontically treated teeth., Aim: The purpose of this study was to assess the prevalence of periapical radiolucencies in a Bulgarian subpopulation and the quality of previous endodontic treatment using CBCT scans., Materials and Methods: This study included 2795 roots from 160 Large FOV CBCT which were evaluated by two independent examiners using two scoring systems: CBCT-PAI and PESS., Results: The inter-examiner agreement spanned from strong to almost perfect (0.892 and 0.983). The prevalence of periapical lesions according to the two scoring systems was 23.1% and 12.9 %, respectively. The prevalence of endodontically treated teeth was high (34.1%). Sixty-five percent of them presented with signs of periapical radiolucencies, while only 1.4% of all non-treated roots had a periapical lesion. A significant association between periapical disease, poor quality of the root canal filling and inadequate coronal seal was found (p<0.001)., Conclusions: The prevalence of periapical disease in endodontically-treated teeth in the Bulgarian subpopulation was high. Poor qual-ity of the root canal filling and inadequate coronal seal were assessed as prognostic determinants of treatment failure. CBCT techniques can augment conventional diagnostic techniques in the field of endodontics., (This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies"., Methods: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation., Results: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi., Conclusions: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis -Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor-coated vesicles at the trans -Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis -Golgi compartment.

The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The quote "bring it back, bring it back, don't take it away from me" from Queen's Love of my life describes the function of the sorting receptor RER1, a 23 kDa protein with four transmembrane domains (TMDs) that localizes to the intermediate compartment and the cis -Golgi. From there it returns escaped proteins that are not supposed to leave the endoplasmic reticulum (ER) back to it. Unique about RER1 is its ability to recognize its ligands through binding motifs in TMDs. Among its substrates are ER-resident proteins, as well as unassembled subunits of multimeric complexes that are retrieved back into the ER, this way guarding the full assembly of their respective complexes. The basic mechanisms for RER1-dependent retrieval have been already elucidated some years ago in yeast. More recently, several important cargoes of RER1 have been described in mammalian cells, and the in vivo role of RER1 is being unveiled by using mouse models. In this Review, we give an overview of the cell biology of RER1 in different models, discuss its controversial role in the brain and provide an outlook on future directions for RER1 research., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Hazara nairovirus (HAZV) is an enveloped trisegmented negative-strand RNA virus classified within the Nairoviridae family of the Bunyavirales order and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly, and egress is poorly understood. Here, we generated a recombinant HAZV expressing enhanced green fluorescent protein and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi apparatus and the endoplasmic reticulum, as well as intra-Golgi transport. We show the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle: an early stage prior to and including gene expression and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titers by approximately 1,000-fold. Treatment of HAZV-infected cells with brefeldin A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed that this late COPI-dependent stage was Arf1 dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1 independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a noncanonical Arf1-independent process suggests that COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport. IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analyzed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi apparatus-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle: an early stage prior to and including gene expression and also a later stage during assembly of infectious virus, with COPI knockdown reducing titers by approximately 1,000-fold. Importantly, while the late stage was Arf1 dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1 independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions and suggest a new Arf1-independent role for components of the COPI coatomer complex., (Copyright © 2020 Fuller et al.)

Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Travis et al.)

Coat protein I (COPI)-coated vesicles mediate retrograde transport from the Golgi to the endoplasmic reticulum (ER), as well as transport within the Golgi. Major progress has been made in defining the structure of COPI coats, in vitro and in vivo, at resolutions as high as 9 Å. Nevertheless, important questions remain unanswered, including what specific interactions stabilize COPI coats, how COPI vesicles recognize their target membranes, and how coat disassembly is coordinated with vesicle fusion and cargo delivery. Here, we use X-ray crystallography to identify a conserved site on the COPI subunit α-COP that binds to flexible, acidic sequences containing a single tryptophan residue. One such sequence, found within α-COP itself, mediates α-COP homo-oligomerization. Another such sequence is contained within the lasso of the ER-resident Dsl1 complex, where it helps mediate the tethering of Golgi-derived COPI vesicles at the ER membrane. Together, our findings suggest that α-COP homo-oligomerization plays a key role in COPI coat stability, with potential implications for the coordination of vesicle tethering, uncoating, and fusion., Competing Interests: The authors declare no competing interest.

Cellular activities, such as growth and secretion, are dependent on correct protein folding and intracellular protein transport. Injury, like ischemia, malnutrition, and invasion of toxic substances, affect the folding environment in the endoplasmic reticulum (ER). The ER senses this information, following which cells adapt their response to varied situations through the unfolded protein response. Activation of the KDEL receptor, resulting from the secretion from the ER of chaperones containing the KDEL sequence, plays an important role in this adaptation. The KDEL receptor was initially shown to be necessary for the retention of KDEL sequence-containing proteins in the ER. However, it has become clear that the activated KDEL receptor also regulates bidirectional transport between the ER and the Golgi complex, as well as from the Golgi to the secretory pathway. In addition, it has been suggested that the signal for KDEL receptor activation may also affect several other cellular activities. In this review, we discuss KDEL receptor-mediated bidirectional transport and signaling and describe disease models and human diseases related to KDEL receptor dysfunction.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly and the leading cause of dementia in humans. Evidence shows that cellular trafficking and recycling machineries are associated with AD risk. A recent study found that the coat protein complex I (COPI)-dependent trafficking in vivo could significantly reduce amyloid plaques in the cortex and hippocampus of neurological in the AD mouse models and identified 12 single-nucleotide polymorphisms in COPI genes to be significantly associated with increased AD risk using 6,795 samples. Here, we used a large-scale GWAS dataset to investigate the potential association between the COPI genes and AD susceptibility by both SNP and gene-based tests. The results showed that only rs9898218 was associated with AD risk with P = 0.017. We further conducted an expression quantitative trait loci (eQTLs) analysis and found that rs9898218 G allele was associated with increased COPZ2 expression in cerebellar cortex with P = 0.0184. Importantly, the eQTLs analysis in whole blood further indicated that 11 of these 12 genetic variants could significantly regulate the expression of COPI genes. Hence, these findings may contribute to understand the association between COPI genes and AD susceptibility., (Copyright © 2019 Yang, Wang, Ju, Sun and Zhang.)

The Golgi apparatus is an important site for the modification of most secreted and membrane proteins. Glycan processing is the major class of modification and is mediated by a large number of Golgi-resident glycosyltransferases and glycosidases. These Golgi enzymes are largely type II transmembrane domain (TMD) proteins consisting of a short N-terminal cytosolic tail, a relatively short TMD and a lumenal 'stem/stalk' region which acts as a spacer between the catalytic domain and the lipid bilayer. The cytosolic tail, TMD, and stem together make what is termed the CTS domain which is responsible for the specific localisation of these enzymes within sub-Golgi compartments via multiple mechanisms. In addition, the catalytic domains of some Golgi enzymes are secreted as a consequence of proteolytic cleavage within their TMDs or stem regions. Finally, there is evidence to suggest that when the retention of Golgi enzymes is perturbed they are targeted for lysosomal degradation., (© 2019 Federation of European Biochemical Societies.)

Intracellular transport is one of the most confusing issues in the field of cell biology. Many different models and their combinations have been proposed to explain the experimental data on intracellular transport. Here, we analyse the data related to the mechanisms of endoplasmic reticulum-to-Golgi and intra-Golgi transport from the point of view of the main models of intracellular transport; namely: the vesicular model, the diffusion model, the compartment maturation-progression model, and the kiss-and-run model. This review initially describes our current understanding of Golgi function, while highlighting the recent progress that has been made. It then continues to discuss the outstanding questions and potential avenues for future research with regard to the models of these transport steps. To compare the power of these models, we have applied the method proposed by K. Popper; namely, the formulation of prohibitive observations according to, and the consecutive evaluation of, previous data, on the basis on the new models. The levels to which the different models can explain the experimental observations are different, and to date, the most powerful has been the kiss-and-run model, whereas the least powerful has been the diffusion model.

A long-standing assumption is that the cisternae of the Golgi apparatus can be grouped into functionally distinct compartments, yet the molecular identities of those compartments have not been clearly described. The concept of a compartmentalized Golgi is challenged by the cisternal maturation model, which postulates that cisternae form de novo and then undergo progressive biochemical changes. Cisternal maturation can potentially be reconciled with Golgi compartmentation by defining compartments as discrete kinetic stages in the maturation process. These kinetic stages are distinguished by the traffic pathways that are operating. For example, a major transition occurs when a cisterna stops producing COPI vesicles and begins producing clathrin-coated vesicles. This transition separates one kinetic stage, the "early Golgi," from a subsequent kinetic stage, the "late Golgi" or " trans -Golgi network (TGN)." But multiple traffic pathways drive Golgi maturation, and the periods of operation for different traffic pathways can partially overlap, so there is no simple way to define a full set of Golgi compartments in terms of kinetic stages. Instead, we propose that the focus should be on the series of transitions experienced by a Golgi cisterna as various traffic pathways are switched on and off. These traffic pathways drive changes in resident transmembrane protein composition. Transitions in traffic pathways seem to be the fundamental, conserved determinants of Golgi organization. According to this view, the initial goal is to identify the relevant traffic pathways and place them on the kinetic map of Golgi maturation, and the ultimate goal is to elucidate the logic circuit that switches individual traffic pathways on and off as a cisterna matures.

Accurate glycosylation of proteins is essential for their function and their intracellular transport. Numerous diseases have been described, where either glycosylation or intracellular transport of proteins is impaired. Coat protein I (COPI) is involved in anterograde and retrograde transport of proteins between endoplasmic reticulum and Golgi, where glycosylation takes place, but no association of defective COPI proteins and glycosylation defects has been described so far. We identified a patient whose phenotype at a first glance was reminiscent of PGM1 deficiency, a disease that also affects N-glycosylation of proteins. More detailed analyses revealed a different disease with a glycosylation deficiency that was only detectable during episodes of acute illness of the patient. Trio-exome analysis revealed a de novo loss-of-function mutation in ARCN1, coding for the delta-COP subunit of COPI. We hypothesize that the capacity of flow through Golgi is reduced by this defect and at high protein synthesis rates, this bottleneck also manifests as transient glycosylation deficiency., (© 2019 Wiley Periodicals, Inc.)

Influenza is an acute transmissible respiratory infectious disease in humans and animals with high morbidity and mortality. It was reported that luteolin, extracted from Chinese herbs, could potently inhibit influenza virus replication in vitro. To assess the effect and explore the fundamental mechanism of luteolin, we infected several cell lines with two subtypes of influenza A virus (IAV), including A/Jiangxi/312/2006 (H3N2) and A/Fort Monmouth/1/1947 (H1N1) and demonstrated that luteolin suppressed the replication of IAV by cytopathic effect reduction method, qRT-PCR, immunofluorescence and Western blot assays. A time-of-addition assay indicated that this compound interfered with viral replication at the early stage of infection. In addition, we found that luteolin suppressed coat protein I complex expression, which was related to influenza virus entry and endocytic pathway. Overall, our findings demonstrated the antiviral effect of luteolin against IAV and its novel antiviral mechanism.

We report here the finding of abnormal Golgi apparatus morphology in motor neuron like cells depleted of SMN as well as Golgi apparatus morphology in SMA patient fibroblasts. Rescue experiments demonstrate that this abnormality is dependent on SMN, but can also be rescued by expression of the COPI coatomer subunit alpha-COP. A motor neuron-like cell line containing an inducible alpha-COP shRNA was created to generate a parallel system to study knockdown of SMN or alpha-COP. Multiple assays of COPI-dependent intracellular trafficking in cells depleted of SMN demonstrate that alpha-COP function is suboptimal, including failed sequestration of plasma membrane proteins, altered binding of mRNA, and defective targeting and transport of Golgi-resident proteins., (Published by Elsevier B.V.)

Background: Protein secretion is an essential process in all eukaryotes including organisms belonging to the phylum Apicomplexa, which includes many intracellular parasites. The apicomplexan parasites possess a specialized collection of secretory organelles that release a number of proteins to facilitate the invasion of host cells and some of these proteins also participate in immune evasion. Like in other eukaryotes, these parasites possess a series of membrane-bound compartments, namely the endoplasmic reticulum (ER), the intermediate compartments (IC) or vesicular tubular clusters (VTS) and Golgi complex through which proteins pass in a sequential and vectorial fashion. Two sets of proteins; COPI and COPII are important for directing the sequential transfer of material between the ER and Golgi complex., Results: Here, using in silico approaches, we identify the components of COPI and COPII complexes in the genome of apicomplexan organisms. The results showed that the COPI and COPII protein complexes are conserved in most apicomplexan genomes with few exceptions. Diversity among the components of COPI and COPII complexes in apicomplexan is either due to the absence of a subunit or due to the difference in the number of protein domains. For example, the COPI epsilon subunit and COPII sec13 subunit is absent in Babesia bovis, Theileria parva, and Theileria annulata genomes. Phylogenetic and domain analyses for all the proteins of COPI and COPII complexes was performed to predict their evolutionary relationship and functional significance., Conclusions: The study thus provides insights into the apicomplexan COPI and COPII coating machinery, which is crucial for parasites secretory network needed for the invasion of host cells.

Intracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on the formation and fusion of vesicular carriers. Coat protein complex (COP) II vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER), whereas COPI vesicles facilitate traffic from the Golgi to the ER and intra-Golgi transport. Mammalian cells express various isoforms of COPII and COPI coat proteins. To investigate the roles of coat protein paralogs, we have combined in vitro vesicle reconstitution from semi-intact cells with SILAC-based mass spectrometric analysis. Here, we describe the core proteomes of mammalian COPII and COPI vesicles. Whereas the compositions of COPII vesicles reconstituted with various isoforms of the cargo-binding subunit Sec24 differ depending on the paralog used, all of the isoforms of the COPI coat produce COPI-coated vesicles with strikingly similar protein compositions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

As a non-ligand-dependent activation protein, EGFRvIII is the most common mutant of EGFR, and its existence or especially its nuclear translocation in tumors can exacerbate the malignancy. Compared with the nuclear translocation of EGFR, which has been studied extensively, the specific mechanism by which EGFRvIII undergoes nuclear translocation has not yet been reported. Here, we found that EGFRvIII eventually reached the nucleus with the involvement of the Golgi and endoplasmic reticulum (ER) in glioma cells. In this process, syntaxin-6 was responsible for the identification and transport of EGFRvIII on Golgi. We also demonstrated that COPI mediated the reverse transport of EGFRvIII from the Golgi to ER, which process was also important for EGFRvIII's nuclear accumulation. EGFRvIII's nuclear translocation can significantly promote STAT3 phosphorylation and PKM2 nuclear localization. Finally, we showed that EGFRvIII's nuclear translocation obviously induced the growth of gliomas in an intracranial xenotransplantation experiment. These data suggested that searching methods that inhibit EGFRvIII entry into the nucleus will be effective glioma treatments., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.