Abstract
Mobilization of Ca2+ from the endoplasmic reticulum (ER) suppresses translational initiation and inhibits post-translational processing and secretion of glycoproteins. This study explores the mechanism whereby ionomycin, a Ca2+ ionophore, and thapsigargin, an ER Ca(2+)-ATPase inhibitor, promote retention of alpha 1-antitrypsin (alpha 1-AT) bearing high mannose, endoglycosidase H (Endo H)-sensitive oligosaccharide side chains within the ER of HepG2 cells. Arrest occurred at the removal of mannose residues such that intermediates with Man7-9GlcNAc2 side chains accumulated with the Man8-9GlcNAc2 structures predominating. Maturation of alpha 1-AT bearing Man5-6GlcNAc2 side chains was unaffected. Inhibition of alpha 1-AT processing by ionomycin occurred independently of translational suppression. Forms of alpha 1-AT identical to those retained with ionomycin or thapsigargin were observed upon treatment with the alpha-1,2-mannosidase inhibitor 1-deoxymannojirimycin whereas castanospermine, an inhibitor of ER alpha-glucosidase I, produced different forms of the glycoprotein. Neither inhibitor impaired transport or secretion of alpha 1-AT. With brefeldin A, which causes redistribution of Golgi enzymes to the ER, alpha 1-AT was retained intracellularly but acquired resistance to Endo H. With ionomycin, thapsigargin, or 1-deoxymannojirimycin-treated cells, however, brefeldin A failed to promote further processing of the glycoprotein. Possible mechanisms for the suppression of alpha 1-AT processing at the alpha-1,2-mannosidase step by Ca(2+)-mobilizing agents are discussed. Excepting tunicamycin, traditional inhibitors of protein processing did not affect amino acid incorporation.
Highlights
This study explores the mechanism whereby viewed as regulatoryforavariety of intracellularevents
Lysates were at concentrations that strongly suppress mRNA translation clarified by centrifugation at 12,000 rpm
Monolayers werewashedtwice with buffered saline, suspended in fresh medium containing unlabeled methionine and each respective drug, and the incubation continued for 60 min (Chase).Additional titrypsin of HepG2 cells (30), this reaction ifsollowed by the removal of two a-1,2-linked mannose residues to produce a Man7GlcNAc2 oligosaccharidestructure
Summary
Monolayers werewashedtwice with buffered saline, suspended in fresh medium containing unlabeled methionine and each respective drug, and the incubation continued for 60 min (Chase).Additional titrypsin of HepG2 cells (30), this reaction ifsollowed by the removal of two a-1,2-linked mannose residues to produce a Man7GlcNAc2 oligosaccharidestructure. Cells incubated for 1 h with or without addition of brefeldin A (5 pg/ml) were pulse-labeled for[10] min with [:"S]methionine and chased in the respective presence or absence of drug.
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