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1.
Nat Commun ; 12(1): 2673, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976123

RESUMO

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Inositol/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Colesterol/metabolismo , Endocitose , Células HEK293 , Células HeLa , Humanos , Inositol Polifosfato 5-Fosfatases/genética , Inositol Polifosfato 5-Fosfatases/metabolismo , Microscopia Confocal , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Triexosilceramidas/metabolismo
2.
Nat Commun ; 12(1): 2034, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795673

RESUMO

COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mapas de Interação de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera
3.
Nat Commun ; 12(1): 1463, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674590

RESUMO

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.


Assuntos
Cinesina/química , Cinesina/metabolismo , Microtúbulos/metabolismo , Trifosfato de Adenosina , Animais , Sítios de Ligação , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinesina/genética , Camundongos , Paclitaxel , Transporte Proteico , Células RAW 264.7
4.
Cells ; 10(3)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652973

RESUMO

Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that-besides traditional ER-Golgi communication-the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen.


Assuntos
Retículo Endoplasmático/virologia , Espaço Extracelular/virologia , Complexo de Golgi/virologia , Membranas Intracelulares/virologia , Via Secretória , Liberação de Vírus , Animais , /virologia , Centrossomo/metabolismo , Espaço Extracelular/metabolismo , Complexo de Golgi/metabolismo , Humanos , Transporte Proteico
5.
Int J Mol Med ; 47(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33537825

RESUMO

The Golgi apparatus is known to underpin many important cellular homeostatic functions, including trafficking, sorting and modifications of proteins or lipids. These functions are dysregulated in neurodegenerative diseases, cancer, infectious diseases and cardiovascular diseases, and the number of disease­related genes associated with Golgi apparatus is on the increase. Recently, many studies have suggested that the mutations in the genes encoding Golgi resident proteins can trigger the occurrence of diseases. By summarizing the pathogenesis of these genetic diseases, it was found that most of these diseases have defects in membrane trafficking. Such defects typically result in mislocalization of proteins, impaired glycosylation of proteins, and the accumulation of undegraded proteins. In the present review, we aim to understand the patterns of mutations in the genes encoding Golgi resident proteins and decipher the interplay between Golgi resident proteins and membrane trafficking pathway in cells. Furthermore, the detection of Golgi resident protein in human serum samples has the potential to be used as a diagnostic tool for diseases, and its central role in membrane trafficking pathways provides possible targets for disease therapy. Thus, we also introduced the clinical value of Golgi apparatus in the present review.


Assuntos
Doença , Complexo de Golgi/metabolismo , Animais , Biomarcadores/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Mutação/genética
6.
Nat Commun ; 12(1): 61, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397928

RESUMO

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Lipoilação , Luciferases/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
7.
Nat Commun ; 12(1): 105, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397973

RESUMO

Environmental triggers have important functions in multiple sclerosis (MS) susceptibility, phenotype, and trajectory. Exposure to early life trauma (ELT) has been associated with higher relapse rates in MS patients; however, the underlying mechanisms are not well-defined. Here we show ELT induces mechanistic and phenotypical alterations during experimental autoimmune encephalitis (EAE). ELT sustains downregulation of immune cell adrenergic receptors, which can be attributed to chronic norepinephrine circulation. ELT-subjected mice exhibit interferon-ß resistance and neurodegeneration driven by lymphotoxin and CXCR2 involvement. These phenotypic changes are observed in control EAE mice treated with ß1 adrenergic receptor antagonist. Conversely, ß1 adrenergic receptor agonist treatment to ELT mice abrogates phenotype changes via restoration of immune cell ß1 adrenergic receptor function. Our results indicate that ELT alters EAE phenotype via downregulation of ß1 adrenergic signaling in immune cells. These results have implications for the effect of environmental factors in provoking disease heterogeneity and might enable prediction of long-term outcomes in MS.


Assuntos
Regulação para Baixo , Interferon beta/metabolismo , Esclerose Múltipla/complicações , Degeneração Neural/complicações , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais , Estresse Psicológico/complicações , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/patologia , Feminino , Complexo de Golgi/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Degeneração Neural/sangue , Degeneração Neural/imunologia , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Norepinefrina/sangue , Fenótipo , Índice de Gravidade de Doença , Regulação para Cima/efeitos dos fármacos
8.
Nat Commun ; 12(1): 614, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504762

RESUMO

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.


Assuntos
Isocitrato Desidrogenase/genética , Mutação/genética , Organelas/metabolismo , Fosfolipídeos/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Glioblastoma/patologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Modelos Biológicos , Oligodendroglioma/patologia , Estearoil-CoA Dessaturase/metabolismo
9.
Int J Mol Sci ; 22(1)2021 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-33401608

RESUMO

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.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator 1 de Ribosilação do ADP/genética , Complexo I de Proteína do Envoltório/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
10.
Nature ; 590(7845): 326-331, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33505018

RESUMO

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.


Assuntos
Glicemia/metabolismo , Antagonistas da Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Animais , Glicemia/análise , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , Clatrina/metabolismo , Células Endócrinas/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Teste de Tolerância a Glucose , Complexo de Golgi/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Proteínas de Neoplasias/química , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia
11.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33452023

RESUMO

Golgins are coiled-coil proteins that play prominent roles in maintaining the structure and function of the Golgi complex. However, the role of golgin proteins in phytopathogenic fungi remains poorly understood. In this study, we functionally characterized the Fusarium graminearum golgin protein RUD3, a homolog of ScRUD3/GMAP-210 in Saccharomyces cerevisiae and mammalian cells. Cellular localization observation revealed that RUD3 is located in the cis-Golgi. Deletion of RUD3 caused defects in vegetative growth, ascospore discharge, deoxynivalenol (DON) production, and virulence. Moreover, the Δrud3 mutant showed reduced expression of tri genes and impairment of the formation of toxisomes, both of which play essential roles in DON biosynthesis. We further used green fluorescent protein (GFP)-tagged SNARE protein SEC22 (SEC22-GFP) as a tool to study the transport between the endoplasmic reticulum (ER) and Golgi and observed that SEC22-GFP was retained in the cis-Golgi in the Δrud3 mutant. RUD3 contains the coiled coil (CC), GRAB-associated 2 (GA2), GRIP-related Arf binding (GRAB), and GRAB-associated 1 (GA1) domains, which except for GA1, are indispensable for normal localization and function of RUD3, whereas only CC is essential for normal RUD3-RUD3 interaction. Together, these results demonstrate how the golgin protein RUD3 mediates retrograde trafficking in the ER-to-Golgi pathway and is necessary for growth, ascospore discharge, DON biosynthesis, and pathogenicity in F. graminearum IMPORTANCE Fusarium head blight (FHB) caused by the fungal pathogen Fusarium graminearum is an economically important disease of wheat and other small grain cereal crops worldwide, and limited effective control strategies are available. A better understanding of the regulation mechanisms of F. graminearum development, deoxynivalenol (DON) biosynthesis, and pathogenicity is therefore important for the development of effective control management of this disease. Golgins are attached via their extreme carboxy terminus to the Golgi membrane and are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. In this study, we systematically characterized a highly conserved Golgin protein, RUD3, and found that it is required for vegetative growth, ascospore discharge, DON production, and pathogenicity in F. graminearum Our findings provide a comprehensive characterization of the golgin family protein RUD3 in plant-pathogenic fungus, which could help to identify a new potential target for effective control of this devastating disease.


Assuntos
Proteínas Fúngicas/fisiologia , Fusarium , Proteínas da Matriz do Complexo de Golgi/fisiologia , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Fusarium/fisiologia , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Filogenia , Doenças das Plantas/microbiologia , Reprodução Assexuada , Esporos Fúngicos , Tricotecenos/metabolismo , Triticum/microbiologia , Virulência
12.
Methods Mol Biol ; 2217: 301-311, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215388

RESUMO

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5ß1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Integrina alfa5beta1/genética , Junções Íntimas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transporte Biológico , Polaridade Celular , Cultura em Câmaras de Difusão , Células Endoteliais/ultraestrutura , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Integrina alfa5beta1/metabolismo , Microscopia de Fluorescência/métodos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Junções Íntimas/ultraestrutura
13.
Traffic ; 22(3): 64-77, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33314495

RESUMO

The endoplasmic reticulum (ER) is involved in biogenesis, modification and transport of secreted and membrane proteins. The ER membranes are spread throughout the cell cytoplasm as well as the export domains known as ER exit sites (ERES). A subpopulation of ERES is centrally localized proximal to the Golgi apparatus. The significance of this subpopulation on ER-to-Golgi transport remains unclear. Transport carriers (TCs) form at the ERES via a COPII-dependent mechanism and move to Golgi on microtubule (MT) tracks. It was shown previously that ERES are distributed along MTs and undergo chaotic short-range movements and sporadic rapid long-range movements. The long-range movements of ERES are impaired by either depolymerization of MTs or inhibition of dynein, suggesting that ERES central concentration is mediated by dynein activity. We demonstrate that the processive movements of ERES are frequently coupled with the TC departure. Using the Sar1a[H79G]-induced ERES clustering at the perinuclear region, we identified BicaudalD2 (BicD2) and Rab6 as components of the dynein adaptor complex which drives perinuclear ERES concentration at the cell center. BicD2 partially colocalized with ERES and with TC. Peri-Golgi ERES localization was significantly affected by inhibition of BicD2 function with its N-terminal fragment or inhibition of Rab6 function with its dominant-negative mutant. Golgi accumulation of secretory protein was delayed by inhibition of Rab6 and BicD2. Thus, we conclude that a BicD2/Rab6 dynein adaptor is required for maintenance of Golgi-associated ERES. We propose that Golgi-associated ERES may enhance the efficiency of the ER-to-Golgi transport.


Assuntos
Retículo Endoplasmático , Complexo de Golgi , Transporte Biológico , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares , Microtúbulos , Transporte Proteico
14.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33156328

RESUMO

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP-OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP-OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport-defective mutant, but not of cholesterol sensing-defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER-Golgi MCSs, depending on the ER cholesterol.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Colesterol/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Transporte Proteico , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
15.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33301566

RESUMO

GRASP55 and GRASP65 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, RNAi or gene knockout approaches to dissect their respective roles have often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for induced rapid degradation by the proteasome. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115, and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, GRASP55 and/or GRASP65 is not required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking but are involved in maintaining the integrity of the Golgi ribbon together with GM130 and Golgin-45.


Assuntos
Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteólise , Brefeldina A/farmacologia , Linhagem Celular , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Ácidos Indolacéticos/farmacologia , Interfase/efeitos dos fármacos , Nocodazol/farmacologia , Proteólise/efeitos dos fármacos
16.
Plant Cell Physiol ; 62(2): 272-279, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33367686

RESUMO

Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screen for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L-heat stress but not to S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2 and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1-1 mutants was similar to that of the wild type (WT). Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the WT. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1) - accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins - were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not by bZIP28, resulting in the initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in the MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER-Golgi vesicle tethering.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático , Genes de Plantas/fisiologia , Complexo de Golgi/fisiologia , Resposta ao Choque Térmico
17.
Exp Cell Res ; 399(2): 112442, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359467

RESUMO

Rab30 is a poorly characterized small GTPase. Here we show that Rab30 is localised primarily to the TGN and recycling endosomes in a range of cell types, including primary neurons; minor levels of Rab30 were also detected throughout the Golgi stack and early endosomes. Silencing of Rab30 resulted in the dispersal of both early and recycling endosomes and TGN compartments in HeLa cells. By analyzing cargo trafficking in Rab30-silenced and Rab30-overexpressing HeLa cells, we demonstrate that Rab30 plays a role in retrograde trafficking of TGN38 from endosomes to the Golgi, but has no apparent role in the endocytic recycling of the transferrin receptor to the plasma membrane. Five interactive partners with Rab30 were identified by pull-down and MS analysis using GFP-tagged Rab30 mutant, Rab30(Q68L). Two of the interactive partners identified were Arf1 and Arf4, known regulators of endosome to TGN retrograde transport. Knockdown of Arf1 and Arf4 results in GFP-Rab30 decorated tubules arising from the recycling endosomes, suggesting association of Rab30 with tubular carriers. Overall our data demonstrates a role for Rab30 in regulating retrograde transport to the TGN and maintenance of endosomal-TGN organization.


Assuntos
Endossomos/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Rede trans-Golgi/genética , Antígenos CD/metabolismo , Endossomos/genética , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico/genética , Receptores da Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Rede trans-Golgi/metabolismo
18.
PLoS Biol ; 18(12): e3001051, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370778

RESUMO

Coordination of cell growth is essential for the development of the brain, but the molecular mechanisms underlying the regulation of glial and neuronal size are poorly understood. To investigate the mechanisms involved in glial size regulation, we used Caenorhabditis elegans amphid sheath (AMsh) glia as a model and show that a conserved cis-Golgi membrane protein eas-1/GOLT1B negatively regulates glial growth. We found that eas-1 inhibits a conserved E3 ubiquitin ligase rnf-145/RNF145, which, in turn, promotes nuclear activation of sbp-1/ SREBP, a key regulator of sterol and fatty acid synthesis, to restrict cell growth. At early developmental stages, rnf-145 in the cis-Golgi network inhibits sbp-1 activation to promote the growth of glia, and when animals reach the adult stage, this inhibition is released through an eas-1-dependent shuttling of rnf-145 from the cis-Golgi to the trans-Golgi network to stop glial growth. Furthermore, we identified long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA), as downstream products of the eas-1-rnf-145-sbp-1 pathway that functions to prevent the overgrowth of glia. Together, our findings reveal a novel and potentially conserved mechanism underlying glial size control.


Assuntos
Ácido Eicosapentaenoico/farmacologia , Complexo de Golgi/metabolismo , Neuroglia/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Tamanho Celular/efeitos dos fármacos , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Insaturados/metabolismo , Complexo de Golgi/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo
19.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374830

RESUMO

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Membrana Celular/metabolismo , Ceramidas/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/genética , Transporte Proteico
20.
Cells ; 9(12)2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33321764

RESUMO

Glycosylation is the most common post-translational modification of proteins; it mediates their correct folding and stability, as well as their transport through the secretory transport. Changes in N- and O-linked glycans have been associated with multiple pathological conditions including congenital disorders of glycosylation, inflammatory diseases and cancer. Glycoprotein glycosylation at the Golgi involves the coordinated action of hundreds of glycosyltransferases and glycosidases, which are maintained at the correct location through retrograde vesicle trafficking between Golgi cisternae. In this review, we describe the molecular machinery involved in vesicle trafficking and tethering at the Golgi apparatus and the effects of mutations in the context of glycan biosynthesis and human diseases.


Assuntos
Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Animais , Glicosilação , Humanos , Estabilidade Proteica , Transporte Proteico
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