Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle

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

“…Cells were washed twice with ice-cold PBS and scraped into ice-cold homogenization buffer [10 mM by guest, on May 12, 2018 www.jlr.org Downloaded from derived from mature DLO within the ER. Recently we reported on a Co 2+ -activated DLODP activity that liberates OSPs from both truncated and mature DLO, but that does not codistribute with DolP-dependent enzymes of the dolichol cycle during density gradient centrifugation of microsomal membranes (18). In fact, the bulk of this DLODP activity distributed similarly to GA-situated galactosyltransferase (UGT) (18).…”

“…Recently, a Co 2+ -activated liver microsomal DLO diphosphatase (DLODP) activity that splits both mature and truncated DLO to yield OSP and DolP has been reported (18). This reaction might enable DolP recycling from unproductive or potentially dangerous DLO intermediates that occur in CDG.…”

“…Recently we reported on a Co 2+ -activated DLODP activity that liberates OSPs from both truncated and mature DLO, but that does not codistribute with DolP-dependent enzymes of the dolichol cycle during density gradient centrifugation of microsomal membranes (18). In fact, the bulk of this DLODP activity distributed similarly to GA-situated galactosyltransferase (UGT) (18). These results prompted us to examine the subcellular sites of OSP production in living cells using drugs that perturb the endomembrane system (Fig.…”

“…This reaction might enable DolP recycling from unproductive or potentially dangerous DLO intermediates that occur in CDG. However, the potential physiological role of this enzyme in DLO regulation remains uncertain because, during density gradient centrifugation, this activity distributes similarly to a marker of the Golgi apparatus (GA), and differently to those of ER-situated DolP-dependent DLO biosynthetic enzymes (18). The complexity of this data prompted us to evaluate the subcellular compartmentalization and membrane topology of OSP production in human hepatocellular HepG2 carcinoma cells treated with drugs that perturb the endomembrane system.…”

“…Then further components were added to give a final reaction mixture of 100 mM MES (pH 5.5), 1 mM CoCl 2 , 0.1% NP-40, 100 M SW, 100 M KIF, 1 mM CST, and 4.2-8.4 g protein in a volume of 50 l. Incubations were carried out at 37°C for 20 min and processed as described in (18).…”