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Kirsten Sandvig - One of the best experts on this subject based on the ideXlab platform.
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induction of direct endosome to endoplasmic reticulum transport in chinese hamster ovary cho cells ldlf with a temperature sensitive defect in ϵ Coatomer Protein ϵ cop
Journal of Biological Chemistry, 2003Co-Authors: Alicia Llorente, Silje Ugland Lauvrak, Bo Van Deurs, Kirsten SandvigAbstract:In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of epsilon-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in epsilon-COP. At the nonpermissive temperature epsilon-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of epsilon-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of epsilon-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of epsilon-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.
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induction of direct endosome to endoplasmic reticulum transport in chinese hamster ovary cho cells ldlf with a temperature sensitive defect in epsilon Coatomer Protein epsilon cop
Journal of Biological Chemistry, 2003Co-Authors: Alicia Llorente, Silje Ugland Lauvrak, Bo Van Deurs, Kirsten SandvigAbstract:Abstract In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of ϵ-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in ϵ-COP. At the nonpermissive temperature ϵ-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of ϵ-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of ϵ-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of ϵ-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.
Alicia Llorente - One of the best experts on this subject based on the ideXlab platform.
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induction of direct endosome to endoplasmic reticulum transport in chinese hamster ovary cho cells ldlf with a temperature sensitive defect in ϵ Coatomer Protein ϵ cop
Journal of Biological Chemistry, 2003Co-Authors: Alicia Llorente, Silje Ugland Lauvrak, Bo Van Deurs, Kirsten SandvigAbstract:In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of epsilon-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in epsilon-COP. At the nonpermissive temperature epsilon-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of epsilon-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of epsilon-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of epsilon-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.
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induction of direct endosome to endoplasmic reticulum transport in chinese hamster ovary cho cells ldlf with a temperature sensitive defect in epsilon Coatomer Protein epsilon cop
Journal of Biological Chemistry, 2003Co-Authors: Alicia Llorente, Silje Ugland Lauvrak, Bo Van Deurs, Kirsten SandvigAbstract:Abstract In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of ϵ-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in ϵ-COP. At the nonpermissive temperature ϵ-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of ϵ-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of ϵ-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of ϵ-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.
Ira Mellman - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Endosome Function in CHO Cells Bearing a Temperature-sensitive Defect in the Coatomer (COPI) Component e
2016Co-Authors: Thomas Kreis, Ira MellmanAbstract:Recent evidence has suggested that subunits of the Coatomer Protein (COPI) complexes are func-tionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit e-COP. In addition to exhibiting condi-tional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associ-ated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive tem-perature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require deliv-ery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of re-ceptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes re-distributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time re-quired for the loss of detectable e-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant pheno-types were also corrected by transfection of wild-type
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inhibition of endosome function in cho cells bearing a temperature sensitive defect in the Coatomer copi component e cop
Journal of Cell Biology, 1997Co-Authors: Elizabeth Daro, David Sheff, Marie Gomez, Thomas E Kreis, Ira MellmanAbstract:Recent evidence has suggested that subunits of the Coatomer Protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit e-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable e-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type e-COP cDNA demonstrating that they directly or indirectly reflected the e-COP defect. Taken together, the results suggest that e-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.
Geoffrey T Swanson - One of the best experts on this subject based on the ideXlab platform.
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intracellular trafficking of ka2 kainate receptors mediated by interactions with Coatomer Protein complex i copi and 14 3 3 chaperone systems
Journal of Biological Chemistry, 2006Co-Authors: Pornpun Vivithanaporn, Sheng Yan, Geoffrey T SwansonAbstract:Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor Proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the Coatomer Protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and β-COP Protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI Proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex Proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 Proteins, which mediate forward trafficking of other integral signaling Proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.
Michael Shtutman - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of truncated human Coatomer Protein complex subunit ζ1 copζ1
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2016Co-Authors: Sergey Lunev, Michael Shtutman, Marije F W Semmelink, Jia Ling Xian, Anna J A Leenders, Alexander Domling, Matthew R GrovesAbstract:The majority of modern anticancer approaches target DNA/Protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human Coatomer Protein complex I (COPI) subunit ζ (Copζ), a component of the coat Protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.
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Tumor-specific silencing of COPZ2 gene encoding Coatomer Protein complex subunit ζ2 renders tumor cells dependent on its paralogous gene COPZ1
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Michael Shtutman, Mirza Baig, Elina Levina, Gregory Hurteau, Chang-uk Lim, Eugenia V. Broude, Mikhail A. Nikiforov, Timothy T. Harkins, C. Steven Carmack, Ye DingAbstract:Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the Coatomer Protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of Coatomer Protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform Coatomer Protein complex ζ2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.
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A subunit of Coatomer Protein complex offers a novel tumor-specific target through a surprising mechanism.
Autophagy, 2011Co-Authors: Michael Shtutman, Igor B. RoninsonAbstract:COPI, a Coatomer Protein complex of secretory vesicles, is involved in Golgi and endoplasmic reticulum traffic and in early endosome maturation. The loss of COPI results in the fragmentation of Golgi, accumulation of immature autophagosomes, inhibition of autophagy, and cell death. Since COPI is required by all cells, it would appear an unlikely target for cancer treatment. However, our recent function-based genomic screen unexpectedly identified a specific COPI subunit, ζ1, as a cancer-specific target. The existing cancer drugs kill only proliferating but not growth-arrested tumor cells, but the depletion of ζ1 induces cell death in both dividing and nondividing tumor cells, while sparing normal cells. The mechanism of this remarkable tumor selectivity turned out to be surprising and heretofore unprecedented.