DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase.
DAD1, the defender against apoptotic cell death, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line. Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST). Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported. Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme. Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48. Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits. DAD1 can be crosslinked to OST48 in intact microsomes with dithiobis(succinimidylpropionate). Crosslinked ribophorin II-OST48 heterodimers, DAD1-ribophorin II-OST48 heterotrimers and DAD1-ribophorin I-ribophorin II-OST48 heterotetramers also were detected. The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.
Kelleher
D J
DJ
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA 01655-0103, USA.
Gilmore
R
R
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents; D019281:Dimerization; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism / Q000648:ultrastructure; D006602:Hexosyltransferases; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D046911:Macromolecular Substances; D008322:Mammals; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013388:Succinimides; D014166:Transferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D051017:Apoptosis Regulatory Proteins; D003432:Cross-Linking Reagents; C083331:DAD1 protein, human; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011506:Proteins; D013388:Succinimides; C034241:ribophorin; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; C011240:dithiobis(succinimidylpropionate)