Brefeldin A promotes the appearance of oligosaccharyl phosphates derived from Glc3Man9GlcNAc2-PP-dolichol within the endomembrane system of HepG2 cells.

Ahmad Massarweh, Michaël Bosco, Soria Iatmanen-Harbi, Clarice Tessier, Laura Amana, Patricia Busca, Isabelle Chantret, Christine Gravier-Pelletier, Stuart E H Moore

Journal of lipid research 2016 Aug

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We reported an oligosaccharide diphosphodolichol (DLO) diphosphatase (DLODP) that generates dolichyl-phosphate and oligosaccharyl phosphates (OSPs) from DLO in vitro. This enzyme could underlie cytoplasmic OSP generation and promote dolichyl-phosphate recycling from truncated endoplasmic reticulum (ER)-generated DLO intermediates. However, during subcellular fractionation, DLODP distribution is closer to that of a Golgi apparatus (GA) marker than those of ER markers. Here, we examined the effect of brefeldin A (BFA), which fuses the GA with the ER on OSP metabolism. In order to increase the steady state level of truncated DLO while allowing formation of mature DLO (Glc3Man9GlcNAc2-PP-dolichol), dolichyl-P-mannose Man7GlcNAc2-PP-dolichol mannosyltransferase was partially downregulated in HepG2 cells. We show that BFA provokes GA endomannosidase trimming of Glc3Man9GlcNAc2-PP-dolichol to yield a Man8GlcNAc2-PP-dolichol structure that does not give rise to cytoplasmic Man8GlcNAc2-P. BFA also strikingly increased OSP derived from mature DLO within the endomembrane system without affecting levels of Man7GlcNAc2-PP-dolichol or cytoplasmic Man7GlcNAc2-P. The BFA-provoked increase in endomembrane-situated OSP is sensitive to nocodazole, and BFA causes partial redistribution of DLODP activity from GA- to ER-containing regions of density gradients. These findings are consistent with BFA-provoked microtubule-dependent GA-to-ER transport of a previously reported DLODP that acts to generate a novel endomembrane-situated OSP population. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Citation

Ahmad Massarweh, Michaël Bosco, Soria Iatmanen-Harbi, Clarice Tessier, Laura Amana, Patricia Busca, Isabelle Chantret, Christine Gravier-Pelletier, Stuart E H Moore. Brefeldin A promotes the appearance of oligosaccharyl phosphates derived from Glc3Man9GlcNAc2-PP-dolichol within the endomembrane system of HepG2 cells. Journal of lipid research. 2016 Aug;57(8):1477-91