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1.
Annu Rev Cell Dev Biol ; 35: 131-168, 2019 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-31399000

RESUMO

Protein coats are supramolecular complexes that assemble on the cytosolic face of membranes to promote cargo sorting and transport carrier formation in the endomembrane system of eukaryotic cells. Several types of protein coats have been described, including COPI, COPII, AP-1, AP-2, AP-3, AP-4, AP-5, and retromer, which operate at different stages of the endomembrane system. Defects in these coats impair specific transport pathways, compromising the function and viability of the cells. In humans, mutations in subunits of these coats cause various congenital diseases that are collectively referred to as coatopathies. In this article, we review the fundamental properties of protein coats and the diseases that result from mutation of their constituent subunits.


Assuntos
Endossomos/química , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Proteínas de Transporte Vesicular/genética , Animais , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/terapia , Humanos , Transporte Proteico , Proteínas de Transporte Vesicular/metabolismo
2.
Annu Rev Biochem ; 86: 637-657, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28471691

RESUMO

Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with ß-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.


Assuntos
Membrana Celular/ultraestrutura , Clatrina/química , Complexo I de Proteína do Envoltório/química , Vesículas Revestidas/ultraestrutura , Células Eucarióticas/ultraestrutura , Proteínas Monoméricas de Ligação ao GTP/química , Transporte Ativo do Núcleo Celular , Membrana Celular/química , Membrana Celular/metabolismo , Clatrina/genética , Clatrina/metabolismo , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Vesículas Revestidas/química , Vesículas Revestidas/metabolismo , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Evolução Molecular , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestrutura , Expressão Gênica , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos
3.
J Cell Sci ; 137(10)2024 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-38770683

RESUMO

Membrane trafficking, a fundamental cellular process encompassing the transport of molecules to specific organelles, endocytosis at the plasma membrane and protein secretion, is crucial for cellular homeostasis and signalling. Cancer cells adapt membrane trafficking to enhance their survival and metabolism, and understanding these adaptations is vital for improving patient responses to therapy and identifying therapeutic targets. In this Review, we provide a concise overview of major membrane trafficking pathways and detail adaptations in these pathways, including COPII-dependent endoplasmic reticulum (ER)-to-Golgi vesicle trafficking, COPI-dependent retrograde Golgi-to-ER trafficking and endocytosis, that have been found in cancer. We explore how these adaptations confer growth advantages or resistance to cell death and conclude by discussing the potential for utilising this knowledge in developing new treatment strategies and overcoming drug resistance for cancer patients.


Assuntos
Carcinogênese , Membrana Celular , Neoplasias , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endocitose , Transporte Proteico , Complexo de Golgi/metabolismo
4.
Hum Mol Genet ; 32(23): 3263-3275, 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-37658769

RESUMO

The COPI coatomer subunit α-COP has been shown to co-precipitate mRNA in multiple settings, but it was unclear whether the interaction with mRNA was direct or mediated by interaction with an adapter protein. The COPI complex often interacts with proteins via C-terminal dilysine domains. A search for candidate RNA binding proteins with C-terminal dilysine motifs yielded Nucleolin, which terminates in a KKxKxx sequence. This protein was an especially intriguing candidate as it has been identified as an interacting partner for Survival Motor Neuron protein (SMN). Loss of SMN causes the neurodegenerative disease Spinal Muscular Atrophy. We have previously shown that SMN and α-COP interact and co-migrate in axons, and that overexpression of α-COP reduced phenotypic severity in cell culture and animal models of SMA. We show here that in an mRNA independent manner, endogenous Nucleolin co-precipitates endogenous α-COP and ε-COP but not ß-COP which may reflect an interaction with the so-called B-subcomplex rather a complete COPI heptamer. The ability of Nucleolin to bind to α-COP requires the presence of the C-terminal KKxKxx domain of Nucleolin. Furthermore, we have generated a point mutant in the WD40 domain of α-COP which eliminates its ability to co-precipitate Nucleolin but does not interfere with precipitation of partners mediated by non-KKxKxx motifs such as the kainate receptor subunit 2. We propose that via interaction between the C-terminal dilysine motif of Nucleolin and the WD40 domain of α-COP, Nucleolin acts an adaptor to allow α-COP to interact with a population of mRNA.


Assuntos
Atrofia Muscular Espinal , Doenças Neurodegenerativas , Animais , Proteína Coatomer/genética , Ligação Proteica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Atrofia Muscular Espinal/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nucleolina
5.
Adv Exp Med Biol ; 1460: 73-95, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39287849

RESUMO

In this chapter, intestinal lipid transport, which plays a central role in fat homeostasis and the development of obesity in addition to the mechanisms of fatty acids and monoacylglycerol absorption in the intestinal lumen and reassembly of these within the enterocyte was described. A part of the resynthesized triglycerides (triacylglycerols; TAG) is repackaged in the intestine to form the hydrophobic core of chylomicrons (CMs). These are delivered as metabolic fuels, essential fatty acids, and other lipid-soluble nutrients, from enterocytes to the peripheral tissues following detachment from the endoplasmic reticulum membrane. Moreover, the attitudes of multiple receptor functions in dietary lipid uptake, synthesis, and transport are highlighted. Additionally, intestinal fatty acid binding proteins (FABPs), which increase the cytosolic flux of fatty acids via intermembrane transfer in enterocytes, and the functions of checkpoints for receptor-mediated fatty acid signaling are debated. The importance of the balance between storage and secretion of dietary fat by enterocytes in determining the physiological fate of dietary fat, including regulation of blood lipid concentrations and energy balance, is mentioned. Consequently, promising checkpoints regarding how intestinal fat processing affects lipid homeostatic mechanisms and lipid stores in the body and the prevention of obesity-lipotoxicity due to excessive intestinal lipid absorption are evaluated. In this context, dietary TAG digestion, pharmacological inhibition of TAG hydrolysis, the regulation of long-chain fatty acid uptake traffic into adipocytes, intracellular TAG resynthesis, the enlargement of cytoplasmic lipid droplets in enterocytes and constitutional alteration of their proteome, CD36-mediated conversion of diet-derived fatty acid into cellular lipid messengers and their functions are discussed.


Assuntos
Absorção Intestinal , Obesidade , Humanos , Obesidade/metabolismo , Animais , Gorduras na Dieta/metabolismo , Gorduras na Dieta/efeitos adversos , Metabolismo dos Lipídeos , Enterócitos/metabolismo , Triglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo
6.
J Integr Plant Biol ; 66(8): 1639-1657, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38888228

RESUMO

Callose, a ß-1,3-glucan plant cell wall polymer, regulates symplasmic channel size at plasmodesmata (PD) and plays a crucial role in a variety of plant processes. However, elucidating the molecular mechanism of PD callose homeostasis is limited. We screened and identified an Arabidopsis mutant plant with excessive callose deposition at PD and found that the mutated gene was α1-COP, a member of the coat protein I (COPI) coatomer complex. We report that loss of function of α1-COP elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme PdBG2. This process is linked to the functions of ERH1, an inositol phosphoryl ceramide synthase, and glucosylceramide synthase through physical interactions with the α1-COP protein. Additionally, the loss of function of α1-COP alters the subcellular localization of ERH1 and GCS proteins, resulting in a reduction of GlcCers and GlcHCers molecules, which are key sphingolipid (SL) species for lipid raft formation. Our findings suggest that α1-COP protein, together with SL modifiers controlling lipid raft compositions, regulates the subcellular localization of GPI-anchored PDBG2 proteins, and hence the callose turnover at PD and symplasmic movement of biomolecules. Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Glucanos , Plasmodesmos , Esfingolipídeos , Plasmodesmos/metabolismo , Glucanos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Esfingolipídeos/metabolismo , Glucosiltransferases/metabolismo , Glucosiltransferases/genética
7.
Protein Expr Purif ; 212: 106358, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37625737

RESUMO

The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and ß'-COPI subunits to bind these membrane protein "clients" for ER retrieval. These "αWD40" and "ß'WD40" domains are structural homologs that demonstrate distinct selectivity for client proteins. However, elucidation of the atomic-level principles of coatomer-client interactions has been challenging due to the tendency of αWD40 domain to undergo aggregation during expression and purification. Here we describe a rapid recombinant production strategy from E. coli, which substantially enhances the quality of the purified αWD40 domain. The αWD40 purification and crystallization are completed within one day, which minimizes aggregation losses and yields a 1.9 Å resolution crystal structure. We demonstrate the versatility of this strategy by applying it to purify the ß'WD40 domain, which yields crystal structures in the 1.2-1.3 Å resolution range. As an alternate recombinant production system, we develop a cost-effective strategy for αWD40 production in human Expi293 cells. Finally, we suggest a roadmap to simplify these protocols further, which is of significance for the production of WD40 mutants prone to rapid aggregation. The WD40 production strategies presented here are likely to have broad applications because the WD40 domain represents one of the largest families of biomolecular interaction modules in the eukaryotic proteome and is critical for trafficking of host as well as viral proteins such as the SARS-CoV-2 spike protein.


Assuntos
COVID-19 , Humanos , Cristalização , Escherichia coli/genética , SARS-CoV-2
8.
Proc Natl Acad Sci U S A ; 117(33): 19994-20003, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747557

RESUMO

The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , MAP Quinase Quinase 3/metabolismo , Neoplasias/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Complexo I de Proteína do Envoltório/genética , Regulação Neoplásica da Expressão Gênica , Genoma , Via de Sinalização Hippo , Humanos , MAP Quinase Quinase 3/genética , Camundongos , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas de Sinalização YAP
9.
Traffic ; 21(3): 274-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31883188

RESUMO

Protein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi-the Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells, the syntaxin SNARE Sed5p oversees membrane fusion events. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the steady-state localization of Sed5p to the Golgi appears to be primarily conformation-based relying on intra-molecular associations between the Habc domain and SNARE-motif while its tribasic COPI-coatomer binding motif plays a role in intra-Golgi retention.


Assuntos
Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Proteínas de Membrana/fisiologia , Proteínas SNARE/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Qa-SNARE/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/metabolismo
10.
Am J Physiol Lung Cell Mol Physiol ; 323(6): L715-L729, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36255715

RESUMO

Human COPA mutations affecting retrograde Golgi-to-endoplasmic reticulum (ER) protein transport cause diffuse alveolar hemorrhage (DAH) and ER stress ("COPA syndrome"). Patients with SLE also can develop DAH. C57BL/6 (B6) mice with pristane-induced lupus develop monocyte-dependent DAH indistinguishable from human DAH, whereas BALB/c mice are resistant. We examined Copa and ER stress in pristane-induced lupus. Copa expression, ER stress, vascular injury, and apoptosis were assessed in mice and COPA was quantified in blood from patients with SLE. Copa mRNA and protein expression were impaired in B6 mice with pristane-induced DAH, but not in pristane-treated BALB/c mice. An ER stress response (increased Hsp5a/BiP, Ddit3/CHOP, Eif2a, and spliced Xbp1) was seen in lungs from pristane-treated B6, but not BALB/c, mice. Resistance of BALB/c mice to DAH was overcome by treating them with low-dose thapsigargin plus pristane. CB6F1 mice did not develop DAH or ER stress, suggesting that susceptibility was recessive. Increased pulmonary expression of von Willebrand factor (Vwf), a marker of endothelial injury, and the chemokine Ccl2 in DAH suggested that pristane promotes lung microvascular injury and monocyte recruitment. Consistent with that possibility, lung endothelial cells and infiltrating bone marrow-derived cells from pristane-treated B6 mice expressed BiP and showed evidence of apoptosis (annexin-V and activated caspase-3 staining). COPA expression also was low in patients with SLE with lung involvement. Pristane-induced DAH may be initiated by endothelial injury, resulting in ER stress, apoptosis of lung endothelial cells, and recruitment of myeloid cells that propagate lung injury. The pathogenesis of DAH in SLE and COPA syndrome may overlap.


Assuntos
Pneumopatias , Lesão Pulmonar , Lúpus Eritematoso Sistêmico , Vasculite , Humanos , Camundongos , Animais , Alvéolos Pulmonares/patologia , Lesão Pulmonar/patologia , Células Endoteliais/patologia , Camundongos Endogâmicos C57BL , Pulmão/patologia , Pneumopatias/patologia , Hemorragia , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Vasculite/patologia , Estresse do Retículo Endoplasmático
11.
J Transl Med ; 20(1): 18, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991628

RESUMO

BACKGROUND: Cervical cancer is the most fatal gynecological carcinoma in the world. It is urgent to explore novel prognostic biomarkers and intervention targets for cervical cancer. METHODS: Through integrated quantitative proteomic strategy, we investigated the protein expression profiles of cervical cancer; 28 fresh frozen tissue samples (11 adenocarcinoma (AC), 12 squamous cell carcinoma (SCC) and 5 normal cervixes (HC)) were included in discover cohort; 45 fresh frozen tissue samples (19 AC, 18 SCC and 8 HC) were included in verification cohort; 140 paraffin-embedded tissues samples of cervical cancer (85 AC and 55 SCC) were used for immunohistochemical evaluation (IHC) of coatomer protein subunit alpha (COPA) as a prognostic biomarker for cervical cancer; how deficiency of COPA affects cell viability and tumorigenic ability of cervical cancer cells (SiHa cells and HeLa cells) were evaluated by cell counting kit-8 and clone formation in vitro. RESULTS: We identified COPA is a potential prognostic biomarker for cervical cancer in quantitative proteomics analysis. By retrospective IHC analysis, we additionally verified the proteomics results and demonstrated moderate or strong IHC staining for COPA is an unfavourable independent prognostic factor for cervical cancer. We also identified COPA is a potential pharmacological intervention target of cervical cancer by a series of in vitro experiments. CONCLUSION: This study is the first to demonstrate that COPA may contribute to progression of cervical cancer. It can serve as a potential prognostic biomarker and promising intervention target for cervical cancer.


Assuntos
Proteína Coatomer , Neoplasias do Colo do Útero , Biomarcadores , Biomarcadores Tumorais/metabolismo , Feminino , Células HeLa , Humanos , Prognóstico , Proteômica , Estudos Retrospectivos , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo
12.
Proc Natl Acad Sci U S A ; 115(36): 8984-8989, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30126980

RESUMO

The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Glucosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Motivos de Aminoácidos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Glucosiltransferases/genética , Complexo de Golgi/genética , Células HEK293 , Células HeLa , Humanos , Mucolipidoses/enzimologia , Mucolipidoses/genética , Mutação de Sentido Incorreto , Domínios Proteicos
13.
J Cell Sci ; 131(5)2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29535154

RESUMO

The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Complexo I de Proteína do Envoltório/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Humanos , Transporte Proteico/genética
14.
Traffic ; 18(9): 604-621, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28696565

RESUMO

Our understanding of protein and lipid trafficking in eukaryotic cells has been challenged by the finding of different forms of compartmentalization and cargo processing in protozoan parasites. Here, we show that, in the absence of a Golgi compartment in Giardia, proteins destined for secretion are directly sorted and packaged at specialized ER regions enriched in COPII coatomer complexes and ceramide. We also demonstrated that ER-resident proteins are retained at the ER by the action of a KDEL receptor, which, in contrast to other eukaryotic KDEL receptors, showed no interorganellar dynamic but instead acts specifically at the limit of the ER membrane. Our study suggests that the ER-exit sites and the perinuclear ER-membranes are capable of performing protein-sorting functions. In our view, the description presented here suggests that Giardia adaptation represents an extreme example of reductive evolution without loss of function.


Assuntos
Retículo Endoplasmático/metabolismo , Giardia lamblia/metabolismo , Complexo de Golgi/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Receptores de Peptídeos/metabolismo
15.
Fungal Genet Biol ; 123: 78-86, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30550852

RESUMO

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.


Assuntos
Aspergillus nidulans/ultraestrutura , Complexo I de Proteína do Envoltório/química , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Aspergillus nidulans/química , Aspergillus nidulans/genética , Transporte Biológico/genética , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/química , Complexo de Golgi/química , Microscopia de Fluorescência , Mutação , Fenótipo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética
16.
Proc Natl Acad Sci U S A ; 113(25): 6916-21, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27298352

RESUMO

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the µ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Sequência de Aminoácidos , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Bovinos , Homologia de Sequência de Aminoácidos
17.
Biochem Biophys Res Commun ; 495(1): 473-480, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29129687

RESUMO

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA.


Assuntos
Apoptose , Proliferação de Células , Proteína Coatomer/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proteína Coatomer/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteômica
18.
Biochem Biophys Res Commun ; 506(3): 463-470, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30352685

RESUMO

Stasimon (also known as Tmem41b) is an evolutionarily conserved transmembrane protein first identified for its contribution to motor system dysfunction in animal models of the childhood neurodegenerative disease spinal muscular atrophy (SMA). Stasimon was shown to be required for normal neurotransmission in the motor circuit of Drosophila larvae and proper development of motor axons in zebrafish embryos as well as to suppress analogous neuronal phenotypes in SMA models of these organisms. However, the subcellular localization and molecular functions of Stasimon are poorly understood. Here, we combined immunoprecipitation with mass spectrometry to characterize the Stasimon interactome in mammalian cells, which reveals association with components of the endoplasmic reticulum (ER), mitochondria, and the COPI vesicle trafficking machinery. Expanding on the interaction results, we used subcellular fractionation studies and super-resolution microscopy to identify Stasimon as an ER-resident protein that localizes at mitochondria-associated ER membranes (MAM), functionally specialized contact sites between ER and mitochondria membranes. Lastly, through characterization of novel knockout mice, we show that Stasimon is an essential gene for mouse embryonic development. Together, these findings identify Stasimon as a novel transmembrane protein component of the MAM with an essential requirement for mammalian development.


Assuntos
Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Animais , Complexo I de Proteína do Envoltório/metabolismo , Humanos , Camundongos , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Células NIH 3T3 , Transporte Proteico
19.
Mol Cell Neurosci ; 79: 53-63, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28041939

RESUMO

The delta opioid receptor (DOPr) is known to be mainly expressed in intracellular compartments. It remains unknown why DOPr is barely exported to the cell surface, but it seems that a substantial proportion of the immature receptor is trapped within the endoplasmic reticulum (ER) and the Golgi network. In the present study, we performed LC-MS/MS analysis to identify putative protein partners involved in the retention of DOPr. Analysis of the proteins co-immunoprecipitating with Flag-DOPr in transfected HEK293 cells revealed the presence of numerous subunits of the coatomer protein complex I (COPI), a vesicle-coating complex involved in recycling resident proteins from the Golgi back to the ER. Further analysis of the amino acid sequence of DOPr identified multiple consensus di-lysine and di-arginine motifs within the intracellular segments of DOPr. Using cell-surface ELISA and GST pulldown assays, we showed that DOPr interacts with COPI through its intracellular loops 2 and 3 (ICL2 and ICL3, respectively) and that the mutation of the K164AK166 (ICL2) or K250EK252 (ICL3) putative COPI binding sites increased the cell-surface expression of DOPr in transfected cells. Altogether, our results indicate that COPI is a binding partner of DOPr and provide a putative mechanism to explain why DOPr is highly retained inside the cells.


Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Sinais Direcionadores de Proteínas , Receptores Opioides delta/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico , Receptores Opioides delta/química
20.
Proc Natl Acad Sci U S A ; 112(46): 14242-7, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26578768

RESUMO

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding ßγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αß'ε-COP B-subcomplex. We present the structure of the C-terminal µ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP µ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.


Assuntos
Proteína Coatomer/química , Saccharomyces cerevisiae/química , Triptofano/química , Motivos de Aminoácidos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Calorimetria Indireta , Catepsina A/química , Catepsina A/genética , Catepsina A/metabolismo , Proteína Coatomer/genética , Proteína Coatomer/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triptofano/genética , Triptofano/metabolismo
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