Brefeldin A promotes the appearance of oligosaccharyl phosphates derived from Glc3Man9GlcNAc2-PP-dolichol within the endomembrane system of HepG2 cells.
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.
INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France Université Pierre et Marie Curie, Paris 6, Paris, France.
Ahmad
A
Massarweh
Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
Michaël
M
Bosco
INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
Soria
S
Iatmanen-Harbi
INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
Clarice
C
Tessier
INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
Laura
L
Amana
Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
Patricia
P
Busca
INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
Isabelle
I
Chantret
Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
Christine
C
Gravier-Pelletier
INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France stuart.moore@inserm.fr.
Stuart E H
SE
Moore
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D016466:CHO Cells; D003412:Cricetulus; D004288:Dolichol Phosphates / Q000378:metabolism; D004286:Dolichols / Q000031:analogs & derivatives* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism
D004288:Dolichol Phosphates; D004286:Dolichols; C520165:Glc3Man9GlcNAc2-PP-Dol; D009844:Oligosaccharides; D010710:Phosphates; C028306:dolichol monophosphate; D020126:Brefeldin A
ALG12-CDG; Golgi apparatus; N-linked glycosylation; endomannosidase; endoplasmic reticulum; glycolipids; isoprenoids; oligosaccharide-diphosphodolichol diphosphatase; phosphatases/lipid