RESUMEN
Coat protein complex I (COPI) vesicles mediate the retrograde transfer of cargo between Golgi cisternae and from the Golgi to the endoplasmic reticulum (ER). However, their roles in the cell cycle and proliferation are unclear. This study shows that TANGO6 associates with COPI vesicles via two transmembrane domains. The TANGO6 N- and C-terminal cytoplasmic fragments capture RNA polymerase II subunit B (RPB) 2 in the cis-Golgi during the G1 phase. COPI-docked TANGO6 carries RPB2 to the ER and then to the nucleus. Functional disruption of TANGO6 hinders the nuclear entry of RPB2, which accumulates in the cytoplasm, causing cell cycle arrest in the G1 phase. The conditional depletion or overexpression of TANGO6 in mouse hematopoietic stem cells results in compromised or expanded hematopoiesis. Our study results demonstrate that COPI vesicle-associated TANGO6 plays a role in the regulation of cell cycle progression by directing the nuclear transfer of RPB2, making it a potential target for promoting or arresting cell expansion.
Asunto(s)
Proteína Coat de Complejo I , Retículo Endoplásmico , Aparato de Golgi , ARN Polimerasa II , Animales , Ratones , Transporte Activo de Núcleo Celular , Proliferación Celular , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , ARN Polimerasa II/metabolismoRESUMEN
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.
Asunto(s)
Complejo 4 de Proteína Adaptadora , Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Animales , Humanos , Chlorocebus aethiops , Virus de la Coriomeningitis Linfocítica/fisiología , Células Vero , Replicación Viral/genética , Complejo 4 de Proteína Adaptadora/metabolismo , Proteína Coat de Complejo IRESUMEN
Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinï¬ammatory signaling.
Asunto(s)
Proteína Coat de Complejo I , Proteína Coatómero , Niño , Humanos , Proteína Coatómero/genética , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Mutación , Síndrome , Aparato de Golgi/genética , Aparato de Golgi/metabolismoRESUMEN
Sphingolipids are components of plant membranes, and their heterogeneous distribution gives different membrane systems distinct properties. For example, glycosyl inositol phosphoceramides (GIPCs), 1 major type of sphingolipids, aggregate in the outer layer of the plasma membrane (PM), as well as in extracellular vesicles (EVs), including the small (30 to 100â nm) EVs termed exosomes. How these sphingolipids are sorted and trafficked is not clear. In this work, we report that Arabidopsis thaliana TETRASPANIN8 (TET8) acts as a sphingolipid carrier and thus regulates the export of GIPCs from the Golgi apparatus. TET8 recognized the coat protein complex I (COPI) subunit γ2-COPI and moved to its proper location in the PM; this recognition required the TET8 C-terminal tail. Deleting the C-terminal tail of TET8 largely restricted its roles in GIPC transport and endosomal trafficking. Further, we show that TET8 affects EV secretion in association with GIPCs. Thus, our findings shed light on GIPC transport and the molecular machinery involved in EV biogenesis.
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Arabidopsis , Exosomas , Arabidopsis/genética , Arabidopsis/metabolismo , Exosomas/metabolismo , Inositol/metabolismo , Esfingolípidos , Proteína Coat de Complejo I/metabolismoRESUMEN
Next year marks one-quarter of a century since the discovery of the so-called COPI-independent pathway, which operates between the Golgi apparatus and the endoplasmic reticulum (ER) in eukaryotic cells. Unlike almost all other intracellular trafficking pathways, this pathway is not regulated by the physical accumulation of multisubunit proteinaceous coat molecules, but instead by the small GTPase Rab6. What also sets it apart from other pathways is that the transport carriers themselves often take the form of tubules, rather than conventional vesicles. In this review, we assess the relevant literature that has accumulated to date, in an attempt to provide a concerted description of how this pathway is regulated. We discuss the possible cargo molecules that are carried in this pathway, and the likely mechanism of Rab6 tubule biogenesis, including how the cargo itself may play a critical role. We also provide perspective surrounding the various molecular motors of the kinesin, myosin and dynein families that have been implicated in driving Rab6-coated tubular membranes long distances through the cell prior to delivering their cargo to the ER. Finally, we also raise several important questions that require resolution, if we are to ultimately provide a comprehensive molecular description of how the COPI-independent pathway is controlled.
Asunto(s)
Retículo Endoplásmico , Aparato de Golgi , Humanos , Células HeLa , Aparato de Golgi/metabolismo , Retículo Endoplásmico/metabolismo , Proteína Coat de Complejo I/metabolismo , Transporte de ProteínasRESUMEN
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 Ca2+ 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 Ca2+ dysregulation, heightened mitophagy, and a decrease in respiratory capacity. Reintroducing MERCS is sufficient to rescue not only mitochondrial distribution and Ca2+ 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.
Asunto(s)
Proteína Coat de Complejo I , Retículo Endoplásmico , Humanos , Retículo Endoplásmico/metabolismo , Proteína Coat de Complejo I/metabolismo , Aparato de Golgi/metabolismo , Mitocondrias/metabolismo , Axones/metabolismoRESUMEN
Membrane contact sites (MCS) make up a crucial route of inter-organelle non-vesicular transport within the cell. Multiple proteins are involved in this process, which includes the ER-resident proteins vesicle associated membrane protein associated protein A and -B (VAPA/B) that form MCS between the ER and other membrane compartments. Currently most functional data on VAP depleted phenotypes have shown alterations in lipid homeostasis, induction of ER stress, dysfunction of UPR and autophagy, as well as neurodegeneration. Literature on concurrent silencing of VAPA/B is still sparse; therefore, we investigated how it affects the macromolecule pools of primary endothelial cells. Our transcriptomics results showed significant upregulation in genes related to inflammation, ER and Golgi dysfunction, ER stress, cell adhesion, as well as Coat Protein Complex-I and -II (COP-I, COP-II) vesicle transport. Genes related to cellular division were downregulated, as well as key genes of lipid and sterol biosynthesis. Lipidomics analyses revealed reductions in cholesteryl esters, very long chain highly unsaturated and saturated lipids, whereas increases in free cholesterol and relatively short chain unsaturated lipids were evident. Furthermore, the knockdown resulted in an inhibition of angiogenesis in vitro. We speculate that ER MCS depletion has led to multifaceted outcomes, which include elevated ER free cholesterol content and ER stress, alterations in lipid metabolism, ER-Golgi function and vesicle transport, which have led to a reduction in angiogenesis. The silencing also induced an inflammatory response, consistent with upregulation of markers of early atherogenesis. To conclude, ER MCS mediated by VAPA/B play a crucial role in maintaining cholesterol traffic and sustain normal endothelial functions.
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Retículo Endoplásmico , Membranas Intracelulares , Humanos , Células Endoteliales de la Vena Umbilical Humana , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Técnicas de Silenciamiento del Gen , Metabolismo , Neovascularización Fisiológica , Colesterol/metabolismo , Esterificación , Lipidómica , Mapas de Interacción de Proteínas , Aparato de Golgi/metabolismo , Proteína Coat de Complejo I/metabolismoRESUMEN
The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.
Asunto(s)
Proteína Coatómero , Proteínas Activadoras de GTPasa , Proteínas de Saccharomyces cerevisiae , Proteína Coat de Complejo I/metabolismo , Proteína Coatómero/metabolismo , Aparato de Golgi/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismoRESUMEN
During entry, human papillomavirus (HPV) traffics from the cell surface to the endosome and then to the trans-Golgi network (TGN) and Golgi apparatus. HPV must transit across the TGN/Golgi and exit these compartments to reach the nucleus to cause infection, although how these steps are accomplished is unclear. Combining cellular fractionation, unbiased proteomics, and gene knockdown strategies, we identified the coat protein complex I (COPI), a highly conserved protein complex that facilitates retrograde trafficking of cellular cargos, as a host factor required for HPV infection. Upon TGN/Golgi arrival, the cytoplasmic segment of HPV L2 binds directly to COPI. COPI depletion causes the accumulation of HPV in the TGN/Golgi, resembling the fate of a COPI binding-defective L2 mutant. We propose that the L2-COPI interaction drives HPV trafficking through the TGN and Golgi stacks during virus entry. This shows that an incoming virus is a cargo of the COPI complex.
Asunto(s)
Proteína Coat de Complejo I , Virus del Papiloma Humano , Infecciones por Papillomavirus , Internalización del Virus , Humanos , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Virus del Papiloma Humano/fisiología , Infecciones por Papillomavirus/virología , Transporte de ProteínasRESUMEN
Skeletal muscle injuries are common, and damaged myofibers are repaired through proliferation and differentiation of muscle satellite cells. GLUT4 is enriched in GLUT4 storage vesicles (GSVs) and plays a crucial role in the maintenance of muscle function. ArfGAP3 regulates the vesicle transport especially for COPI coat assembly, but its effects on GSVs and the repair process after skeletal muscle injury remains unclear. In this study, datasets related to skeletal muscle injury and myoblast differentiation GSE469, GSE5413, GSE45577 and GSE108040 were retrieved through the GEO database and the expression of heptameric coat protein complex I (COPI) and Golgi vesicle transport-related genes in various datasets, as well as the expression correlation between ArfGAP2, ArfGAP3 and COPI-related genes COPA, COPB1, COPB2, COPE, COPZ1, COPZ2 were analyzed. The results suggested that ArfGAP3 was expressed in the process of repair after skeletal muscle injury and myoblast differentiation and that ArfGAP3 was positively correlated with COPI-related genes. In vitro experimental results showed that ArfGAP3 was colocalized with COPA, COPB, COPG protein, and GLUT4 in C2C12 myoblasts. After the downregulation of ArfGAP3 expression, intracellular vesicle transport, and glucose uptake were blocked, the proliferation of myoblasts under low glucose culture conditions was impaired, the proportion of apoptosis increased, and myotube differentiation was impaired. In summary, ArfGAP3 regulates COPI-associated vesicle and GSVs transport and affects the proliferation and differentiation ability of myoblasts by influencing glucose uptake, thereby modulating the repair process after skeletal muscle injury.
Asunto(s)
Proteína Coat de Complejo I , Proteínas Activadoras de GTPasa , Mioblastos , Transporte Biológico , Diferenciación Celular , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Glucosa/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas Activadoras de GTPasa/metabolismoRESUMEN
The Golgi apparatus is an organelle that mediates modifications, sorting, and transport of proteins and lipids. Golgins are a group of proteins with coiled-coil structures that localize to the Golgi and are thought to function as tethers to facilitate the docking of vesicles, Rab GTPases, and cytoskeleton components to the Golgi stack. Giantin is the longest golgin and has been thought to function as a tether for COPI vesicles along with other golgins, such as p115 and GM130. Contrary to our expectation that the loss of the tether will result in an increase in untethered COPI vesicles in the cytoplasm, our electron microscopy observations showed that the fenestrae normally present in Golgi cisternae were reduced upon Giantin knockdown. We also found that this structural change is accompanied by altered secretion of cargo proteins and cell surface glycosylation. These results indicate that there exists a correlation between Golgi structural changes caused by the loss of Giantin and Golgi function. Here, we describe electron tomography methods for the detection of structural changes in the Golgi.
Asunto(s)
Tomografía con Microscopio Electrónico , Electrones , Proteínas de la Matriz de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Autoantígenos/metabolismo , Aparato de Golgi/metabolismo , Proteína Coat de Complejo I/metabolismoRESUMEN
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.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento , Aparato de Golgi , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Aparato de Golgi/metabolismo , Retículo Endoplásmico/metabolismo , Proteína Coat de Complejo I/metabolismo , LípidosRESUMEN
Bone mineralization is a sophisticated regulated process composed of crystalline calcium phosphate and collagen fibril. Autophagy, an evolutionarily conserved degradation system, whereby double-membrane vesicles deliver intracellular macromolecules and organelles to lysosomes for degradation, has recently been shown to play an essential role in mineralization. However, the formation of autophagosomes in mineralization remains to be determined. Here, we show that Coat Protein Complex I (COPI), responsible for Golgi-to-ER transport, plays a pivotal role in autophagosome formation in mineralization. COPI vesicles were increased after osteoinduction, and COPI vesicle disruption impaired osteogenesis. Mechanistically, COPI regulates autophagy activity via the mTOR complex 1 (mTORC1) pathway, a key regulator of autophagy. Inhibition of mTOR1 rescues the impaired osteogenesis by activating autophagy. Collectively, our study highlights the functional importance of COPI in mineralization and identifies COPI as a potential therapeutic target for treating bone-related diseases.
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Enfermedades Óseas , Calcinosis , Humanos , Autofagia , Vesícula , Lisosomas , Proteína Coat de Complejo I , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
The coatomer protein complex 1 (COPI) is a multisubunit complex that coats intracellular vesicles and is involved in intracellular protein trafficking. Recently we and others found that depletion of COPI complex subunits zeta (COPZ1) and delta (ARCN1) preferentially kills tumor cells relative to normal cells. Here we delineate the specific cellular effects and sequence of events of COPI complex depletion in tumor cells. We find that this depletion leads to the inhibition of mitochondrial oxidative phosphorylation and the elevation of reactive oxygen species (ROS) production, followed by accumulation of lipid droplets (LDs) and autophagy-associated proteins LC3-II and SQSTM1/p62 and, finally, apoptosis of the tumor cells. Inactivation of ROS in COPI-depleted cells with the mitochondrial-specific quencher, mitoquinone mesylate, attenuated apoptosis and markedly decreased both the size and the number of LDs. COPI depletion caused ROS-dependent accumulation of LC3-II and SQSTM1 which colocalizes with LDs. Lack of double-membrane autophagosomes and insensitivity to Atg5 deletion suggested an accumulation of a microlipophagy complex on the surface of LDs induced by depletion of the COPI complex. Our findings suggest a sequence of cellular events triggered by COPI depletion, starting with inhibition of oxidative phosphorylation, followed by ROS activation and accumulation of LDs and apoptosis.
Asunto(s)
Autofagia , Neoplasias , Especies Reactivas de Oxígeno , Apoptosis , Proteína Coat de Complejo I/metabolismo , Lípidos , Neoplasias/metabolismoRESUMEN
Light signaling precisely controls photomorphogenic development in plants. PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and PIF5) play critical roles in the regulation of this developmental process. In this study, we report CONSTITUTIVELY PHOTOMORPHOGENIC 1 SUPPRESSOR 6 (CSU6) functions as a key regulator of light signaling. Loss of CSU6 function largely rescues the cop1-6 constitutively photomorphogenic phenotype. CSU6 promotes hypocotyl growth in the dark, but inhibits hypocotyl elongation in the light. CSU6 not only associates with the promoter regions of PIF4 and PIF5 to inhibit their expression in the morning, but also directly interacts with both PIF4 and PIF5 to repress their transcriptional activation activity. CSU6 negatively controls a group of PIF4- and PIF5-regulated gene expressions. Mutations in PIF4 and/or PIF5 are epistatic to the loss of CSU6, suggesting that CSU6 acts upstream of PIF4 and PIF5. Taken together, CSU6 promotes light-inhibited hypocotyl elongation by negatively regulating PIF4 and PIF5 transcription and biochemical activity.
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Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Hipocótilo/metabolismo , Fitocromo/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Factor V/genética , Factor V/metabolismo , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
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 ß'-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.
Asunto(s)
Proteína Coat de Complejo I/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína Coatómero/genética , Proteína Coatómero/metabolismo , Aparato de Golgi/metabolismo , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.
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Proteínas de Arabidopsis , Arabidopsis , MicroARNs , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteína Coat de Complejo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Guanina/metabolismo , MicroARNs/metabolismo , Nucleótidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mild cognitive impairment (MCI) is a prodromal stage of memory impairment that may precede dementia. MCI is classified by the presence or absence of memory impairment into amnestic or non-amnestic MCI, respectively. More than 90% of patients with amnestic MCI who progress towards dementia meet criteria for Alzheimer's disease (AD). A combination of mechanisms promotes MCI, including intracellular neurofibrillary tangle formation, extracellular amyloid deposition, oxidative stress, neuronal loss, synaptodegeneration, cholinergic dysfunction, cerebrovascular disease, and neuroinflammation. However, emerging evidence indicates that neuroinflammation plays an important role in the pathogenesis of cognitive impairment. Unfortunately, there are currently no Food and Drug Administration (FDA)-approved drugs for MCI. Copolymer-1 (Cop-1), also known as glatiramer acetate, is a synthetic polypeptide of four amino acids approved by the FDA for the treatment of relapsing-remitting multiple sclerosis. Cop-1 therapeutic effect is attributed to immunomodulation, promoting a switch from proinflammatory to anti-inflammatory phenotype. In addition to its anti-inflammatory properties, it stimulates brain-derived neurotrophic factor (BDNF) secretion, a neurotrophin involved in neurogenesis and the generation of hippocampal long-term potentials. Moreover, BDNF levels are significantly decreased in patients with cognitive impairment. Therefore, Cop-1 immunization might promote synaptic plasticity and memory consolidation by increasing BDNF production in patients with MCI.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Factor Neurotrófico Derivado del Encéfalo , Proteína Coat de Complejo I , Progresión de la Enfermedad , Acetato de Glatiramer/uso terapéutico , Humanos , Trastornos de la Memoria , Pruebas NeuropsicológicasRESUMEN
Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach.
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Leucocitos Mononucleares , Nucleotidiltransferasas , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteína Coat de Complejo I/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de SeñalRESUMEN
Positive-stranded RNA viruses modify host organelles to form replication organelles (ROs) for their own replication. The enteroviral 3A protein has been demonstrated to be highly associated with the COPI pathway, in which factors operate on the ER-to-Golgi intermediate and the Golgi. However, Sar1, a COPII factor exerting coordinated action at endoplasmic reticulum (ER) exit sites rather than COPI factors, is required for the replication of foot-and-mouth disease virus (FMDV). Therefore, further understanding regarding FMDV 3A could be key to explaining the differences and to understanding FMDV's RO formation. In this study, FMDV 3A was confirmed as a peripheral membrane protein capable of modifying the ER into vesicle-like structures, which were neither COPII vesicles nor autophagosomes. When the C-terminus of 3A was truncated, it was located at the ER without vesicular modification. This change was revealed using mGFP and APEX2 fusion constructs, and observed by fluorescence microscopy and electron tomography, respectively. For the other 3A truncation, the minimal region for modification was aa 42-92. Furthermore, we found that the remodeling was related to two COPII factors, Sar1 and Sec12; both interacted with 3A, but their binding domains on 3A were different. Finally, we hypothesized that the N-terminus of 3A would interact with Sar1, as its C-terminus simultaneously interacted with Sec12, which could possibly enhance Sar1 activation. On the ER membrane, active Sar1 interacted with regions of aa 42-59 and aa 76-92 from 3A for vesicle formation. This mechanism was distinct from the traditional COPII pathway and could be critical for FMDV RO formation.