Morphogenetic effects of Brefeldin A on embryogenic cell cultures of Daucus carota L.
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Brefeldin A
Endomembrane system
Daucus carota
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Daucus carota
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Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
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The effect of brefeldin A (BFA) on total and polarized protein secretion was examined in MDCK cells.Increasing concentrations of BFA have increasingly inhibitory effects on total protein secretion.The total protein secretion was essentially unaffected by BFA at 0.5 pg/ml.When the BFA concentration was increased to 10 and 30 pg/ml, the total protein secretion was reduced to about 70 and 25%, respectively, of the control level.Consistent with this effect on total protein secretion, the Golgi structure as revealed by C6-NBD-ceramide (a fluorescent ceramide analog) staining was essentially unaltered by 0.5 pg/ml BFA, while 10 and 30 pg/ml BFA significantly dispersed the Golgi apparatus.When the polarity of protein secretion was examined, it was found that the ratio of proteins se- creted from the apical to those from the basolateral surface was reduced from 1.5-2.0 to 0.4-0.7 by all three BFA concentrations.Furthermore, several pro- teins which are preferentially released from the apical surface were found to be released without apparent surface polarity, while several other proteins which were preferentially released from the basolateral surface were unaffected.This study suggests that BFA, at 0.5 pg/ml, can selectively inhibit protein secretion from the apical surface without affecting total protein secretion.The inhibition of apical secretion results in enhanced protein secretion from the basolateral surface.The plasma membrane of epithelial cells is differentiated into morphologically, functionally, and biochemically distinct apical and basolateral domains.Epithelial cells are capable of delivering different membrane and secretory proteins to these two membrane domains(1-4).In MDCK' epithelial cells,
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Effects of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA) on endomembrane structures and lipid synthesis were compared in maize root cells and tobacco Bright Yellow-2 cells. Immunofluorescence and electron microscopy studies showed that NDGA altered the structure and distribution of the endoplasmic reticulum (ER) within 1 h but not of the Golgi apparatus whereas, as shown previously, BFA altered that organization of the Golgi apparatus and, only subsequently, of the ER. Biochemical studies revealed that both drugs and especially BFA led to a strong inhibition of the phytosterol biosynthetic pathway: BFA led to accumulation of sterol precursors. The importance of phytosterols in membrane architecture and membrane trafficking is discussed.
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In this study, we found that Cysteine‐rich with EGF‐like domains 2 (CRELD2), a novel endoplasmic reticulum stress‐inducible protein, is not only localized in the ER‐Golgi apparatus but also spontaneously secreted. Deletion of four C‐terminal amino acids from mouse CRELD2 or addition of tag‐peptides to its C‐terminus dramatically enhanced CRELD2 secretion. Intra‐ and extra‐cellular CRELD2 is differentially glycosylated and its spontaneous secretion was significantly prevented by overexpression of a dominant negative mutant Sar1 and treatment with brefeldin A. Overexpression of wild‐type GRP78 remarkably enhanced the secretion of wild‐type but not mutant CRELD2. Our results demonstrate both that CRELD2 is a novel secretory glycoprotein regulated by Sar1 and GRP78 and that the C‐terminal of CRELD2 plays a crucial role in its secretion.
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Daucus carota
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