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Wntless (Wls) transports Wnt molecules for secretion; however, the cellular mechanism underlying the initial assembly of Wnt secretory vesicles is still not fully defined. Here, we performed proteomic and mutagenic analyses of mam...
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Wntless (Wls) transports Wnt molecules for secretion; however, the cellular mechanism underlying the initial assembly of Wnt secretory vesicles is still not fully defined. Here, we performed proteomic and mutagenic analyses of mammalian Wls, and report a mechanism for formation of early Wnt secretory vesicles on ER membrane. Wls forms a complex with SEC12 (also known as PREB), an ER membrane-localized guanine nucleotide-exchange factor (GEF) activator of the SAR1 (the SAR1A isoform) small GTPase. Compared to palmitoylation-deficient Wnt molecules, binding of mature Wnt to Wls increases Wls–SEC12 interaction and promotes association of Wls with SAR1, the key activator of the COPII machinery. Incorporation of Wls into this exporting ER compartment is affected by Wnt ligand binding and SEC12 binding to Wls, as well as the structural integrity and, potentially, the folding of the cytosolic tail of Wls. In contrast, Wls–SEC12 binding is stable, with the interacting interface biochemically mapped to cytosolic segments of individual proteins. Mutant Wls that fails to communicate with the COPII machinery cannot effectively support Wnt secretion. These data suggest that formation of early Wnt secretory vesicles is carefully regulated to ensure proper export of functional ligands.
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Nutrient transporters have been shown to translocate to the plasma membrane (PM) of the filamentous fungus Aspergillus nidulans via an unconventional trafficking route that bypasses the Golgi. This finding strongly suggests the ex...
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Nutrient transporters have been shown to translocate to the plasma membrane (PM) of the filamentous fungus Aspergillus nidulans via an unconventional trafficking route that bypasses the Golgi. This finding strongly suggests the existence of distinct COPII vesicle subpopulations, one following Golgi-dependent conventional secretion and the other directed towards the PM. Here, we address whether Golgi-bypass concerns cargoes other than nutrient transporters and whether Golgi-bypass is related to cargo structure, size, abundance, physiological function, or polar vs. non-polar distribution in the PM. To address these questions, we followed the dynamic subcellular localization of two selected membrane cargoes differing in several of the aforementioned aspects. These are the proton-pump ATPase PmaA and the PalI pH signaling component. Our results show that neosynthesized PmaA and PalI are translocated to the PM via Golgi-bypass, similar to nutrient transporters. In addition, we showed that the COPII-dependent exit of PmaA from the ER requires the alternative COPII coat subunit LstA, rather than Sec24, whereas PalI requires the ER cargo adaptor Erv14. These findings strengthen the evidence of distinct cargo-specific COPII subpopulations and extend the concept of Golgi-independent biogenesis to essential transmembrane proteins, other than nutrient transporters. Overall, our findings point to the idea that Golgi-bypass might not constitute a fungal-specific peculiarity, but rather a novel major and cargo-specific sorting route in eukaryotic cells that has been largely ignored.
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SelenoproteinK (SelK), an endoplasmic reticulum (ER) - resident protein, possesses the property of mediate oxidation resistance and ER - associated protein degradation (ERAD) in several tissues. Here, we found that increased SelK ...
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SelenoproteinK (SelK), an endoplasmic reticulum (ER) - resident protein, possesses the property of mediate oxidation resistance and ER - associated protein degradation (ERAD) in several tissues. Here, we found that increased SelK markedly promotes fatty acid translocase (CD36) subcellular trafficking and aggravates lipid accumulation in hepatocytes. We demonstrated that SelK is required for the assembly of COPII vesicles and accelerates transport of palmitoylated-CD36 from the ER to Golgi, thus facilitating CD36 plasma membrane distribution both in vivo and in vitro . The mechanism is that SelK increases the stability of Sar1B and triggers CD36-containing nascent COPII vesicle formation, consequently, promotes CD36 subcellular trafficking. Furthermore, we verified that the intervention of SelK SH3 binding domain can inhibit the vesicle formation and CD36 subcellular trafficking, significantly ameliorates NAFLD in mice. Collectively, our findings disclose an unexpected role of SelK in regulating NAFLD development, suggesting that targeting the SelK of hepatocytes may be a new therapeutic strategy for the treatment of NAFLD.
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Aceast? cercetare s-a concentrat pe explorarea modului ?n care introducerea cursurilor de robotic? ?n ?coal? poate ?mbun?t??i procesul instructiv-educativ, ?n special ?n direc?ia antren?rii copiilor c?tre abilit??ile specifice sec...
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Aceast? cercetare s-a concentrat pe explorarea modului ?n care introducerea cursurilor de robotic? ?n ?coal? poate ?mbun?t??i procesul instructiv-educativ, ?n special ?n direc?ia antren?rii copiilor c?tre abilit??ile specifice secolului 21, dar ?i datorit? roboticii care atrage din ce ?n ce mai mult. La aceast? cercetare au participat dou? grupuri de elevi de la o ?coal? de stat din Bucure?ti: ?coala Gimnazial? Nr. 195. Grupul experimental a participat la cursul de Robotic? ?i grupul de control a participat la un curs de Matematic? Aplicat? ?n Transmiterea Mi?c?rii, pe o perioad? de ?apte s?pt?mani, ?n anul 2017. ?n momentul respectiv, elevii din ambele grupuri erau clasa a III-a, iar media lor de varst? era de 9-10 ani. Grupul care a desf??urat cursul de Robotic? a fost compus din 33 elevi, iar grupul care a desf??urat cursul de Matematic? Aplicat? ?n Transmiterea Mi?c?rii a fost compus din 37 elevi. Pentru aceast? cercetare s-au utilizat kiturile furnizate de compania LEGO de tipul LEGO Mindstorm, acestea permi?and elevilor lucrul ?n echip?. Astfel, s-a facilitat ?n?elegerea no?iunilor de baz?, iar copiii au reu?it ulterior s?-?i exprime ?i s?-?i dezvolte ideile proprii. No?iunile de robotic?, conceptele tehnologice de baz? au fost u?or asimilate de grupele de elevi cu media de varst? 10 ani. Dezbaterile dintre elevi, rezolvarea problemelor matematice, munca ?n echip?, valorificarea interesului pe care copiii ?l aveau pentru designul robo?ilor, demonstreaz? c? robotica este o disciplin? ce ca?tig? teritoriu.
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Cargo proteins exported from the endoplasmic reticulum to the Golgi apparatus are typically transported in coat protein complex II (COPII)-coated vesicles of 60-90 nm diameter. Several cargo molecules including collagens and chylo...
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Cargo proteins exported from the endoplasmic reticulum to the Golgi apparatus are typically transported in coat protein complex II (COPII)-coated vesicles of 60-90 nm diameter. Several cargo molecules including collagens and chylomicrons form structures that are too large to be accommodated by these vesicles, but their secretion still requires COPII proteins. Here, we first review recent progress on large cargo secretions derived especially from animal models and human diseases, which indicate the importance of COPII proteins. We then discuss the recent isolation of specialized factors that modulate the process of COPII-dependent cargo formation to facilitate the exit of large-sized cargoes from the endoplasmic reticulum. Based on these findings, we propose a model that describes the importance of the GTPase cycle for secretion of oversized cargoes. Next, we summarize reports that describe the structures of COPII proteins and how these results provide insight into the mechanism of assembly of the large cargo carriers. Finally, we discuss what issues remain to be solved in the future.
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Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic retic...
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Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPIIdependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture.
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The COPII coat and the small GTPase Sar1 mediate protein export from the endoplasmic reticulum (ER) via specialized domains known as the ER exit sites. The peripheral ER protein Sec16 has been proposed to organize ER exit sites. H...
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The COPII coat and the small GTPase Sar1 mediate protein export from the endoplasmic reticulum (ER) via specialized domains known as the ER exit sites. The peripheral ER protein Sec16 has been proposed to organize ER exit sites. However, it remains unclear how these molecules drive COPII coat polymerization. Here, we characterized the spatiotemporal relationships between the Saccharomyces cerevisiae COPII components during their polymerization by performing fluorescence microscopy of an artificial planar membrane. We demonstrated that Sar1 dissociates from the membrane shortly after the COPII coat recruitment, and Sar1 is then no longer required for the COPII coat to bind to the membrane. Furthermore, we found that Sec16 is incorporated within the COPII-cargo clusters, and that this is dependent on the Sar1 GTPase cycle. These data show how Sar1 drives the polymerization of COPII coat and how Sec16 is spatially distributed during COPII coat polymerization.
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Abstract The infection of CHIKV is associated with cellular membranes; however whether early secretory pathways are involved in CHIKV replication remains unclear. In the present study, we have provided initial evidences that CHIKV...
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Abstract The infection of CHIKV is associated with cellular membranes; however whether early secretory pathways are involved in CHIKV replication remains unclear. In the present study, we have provided initial evidences that CHIKV requires both COPI and COPII for its replication. Small interfering RNAs against COPI components, including coatomer, ARFs or GBF1, suppress CHIKV replication. Moreover, CHIKV infection is abolished by the presence of ARF1 inhibitor brefeldin A or GBF1 inhibitor golgicide A. In addition, perturbation of COPII by silencing key components of COPII pathways leads to a reduction in CHIKV replication. Collectively, these observations demonstrate the importance of functional secretory pathways in the infectivity of CHIKV. Highlights ? Coatomer is critical for CHIKV replication. ? Three classes of ARFs are important for CHIKV infection. ? GBF1 is crucial for CHIKV replication. ? Perturbation of COPII components reduces CHIKV infection.
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