DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase

Abstract

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 ref lects the essential nature of N-linked glycosylation in eukaryotes.In multicellular organisms, programmed cell death or apoptosis is an obligatory, exquisitely regulated event during normal cell differentiation, development, and tissue homeostasis of the mature organism (1). During the past several years, it has been established that regulatory proteins (e.g., ced9 and bcl-2) and enzymatic activities [e.g., ced3 and interleukin 1-␤-converting enzyme (ICE)-like proteases] that control commitment to, and mediate progression of, the programmed cell death pathway are conserved between invertebrates and vertebrates (2, 3). The DAD1 protein, the defender against apoptotic cell death, was identified as a mammalian cell death suppressor that may act downstream of the bcl-2 protein (4, 5). A human cDNA encoding a novel 113-residue hydrophobic protein designated as DAD1 was cloned by complementation of the restrictive phenotype of tsBN7 cells, a temperaturesensitive derivative of BHK21 cells, which undergo apoptosis at 37ЊC (4). A point mutation in the DAD1 gene introduces a charged residue within the first hydrophobic segment of the DAD1 protein. Rapid loss of the DAD1 protein from tsBN7 cells after shift to the restrictive temperature initiates apoptosis either because DAD1 acts as a cell death suppressor (4) or because DAD1 performs an essential cellular function, the loss of which leads to programmed cell death. The isolation of a Xenopus laevis DAD1 cDNA that predicted a protein that was 91% identical in sequence to human DAD1 revealed substantial evolutionary conservation (4), an observation that has s...

Function and Factor Interactions of a Locus Control Region Element in the Mouse T Cell Receptor-α/Dad1 Gene Locus

“…In mature T cells, TCR signaling initiates immune responses. Dad1 was initially described as an antiapoptosis gene (2) and was subsequently found to be homologous to a subunit of mammalian oligosaccharyl-transferase (3). Germline deletion of Dad1 results in an embryonic lethal phenotype (4 -6).…”

“…A locus control region (LCR) 3 is a powerful cis-acting control element that can transfer nearly all the transcriptional characteristics of its locus of origin onto a heterologous transgene (9,10). Furthermore, an LCR can accomplish this at any position of integration in the genome of mice.…”

Physical map of a 1.5 Mb region on 12p11.2 harbouring a synpolydactyly associated chromosomal breakpoint

“…Its homolog DAD1 was first isolated in 1993 by Nakashima and co-workers as a defender against apoptotic cell death, 19 and was subsequently shown to be a subunit of oligosaccharyltransferase. 20 Interestingly, some heterozygous Dad1-deficient mice have been reported to display soft-tissue syndactyly, making its homolog DAD-R a likely positional as well as functional candidate gene. 21 KRAG consists of five exons spanning about 20 kb of genomic DNA and encodes a protein which is probably involved in the regulation of cell proliferation.…”

Tansley Review No. 118

“…Using the animal system as a model, homologues to the defender against apoptotic death (dad) gene have been discovered in pea and maize (Gray et al, 1997 ;Orzaez & Granell, 1997). The presence of this gene in both animals and plants is not surprising, as this gene has been shown to code for an oligosaccharide transferase (Kelleher & Gilmore, 1997) essential for glycoprotein synthesis. However, the functional significance of dad, and glycosylation, in cell death in plants remains to be elucidated.…”