The highly conserved DAD1 protein involved in apoptosis is required for N-linked glycosylation

Tomoko Makishiman, Torahiko Nakashima, Kazue Nagata-Kuno, Kohtaro Fukushima, Hiroshi Iida, Masao Sakaguchi, Yukio Ikehara, Sohtaro Komiyama, Takeharu Nishimoto

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

Background: The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation which have been isolated from the BHK21 cell line derived from the golden hamster. It has a mutation in the DAD1 gene encoding a 12.5kDa highly conserved protein through evolution, and enters apoptosis at the restrictive temperature due to this mutation. Results: DAD1 was recovered in light membrane fractions after differential centrifugation. It could not be released from the membrane, even by carbonate extraction, without a detergent. Upon digestion with proteinase K, both N and C terminal portions - but not the middle portions of DAD1 were released from the membrane. Thus, DAD1 appears to be an integral membrane protein in which both termini are located in the cytosol. DAD1 was localized in the endoplasmic reticulum. In accordance with a similarity to the yeast protein Ost2p, which is a subunit of the oligosaccharyl-transferase, at the restrictive temperature, loss of DAD1 function caused a defect of N-linked glycosylation in tsBN7 cells resulting in apoptosis. However, tunicamycin, which is known to inhibit N-linked glycosylation did not induce apoptosis in either tsBN7 or BHK21 cells. Conclusion: tsBN7 cells have a defect in N-linked glycosylation caused by the loss of DAD1.

Original languageEnglish (US)
Pages (from-to)129-141
Number of pages13
JournalGenes to Cells
Volume2
Issue number2
StatePublished - Dec 1 1997
Externally publishedYes

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

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    Makishiman, T., Nakashima, T., Nagata-Kuno, K., Fukushima, K., Iida, H., Sakaguchi, M., Ikehara, Y., Komiyama, S., & Nishimoto, T. (1997). The highly conserved DAD1 protein involved in apoptosis is required for N-linked glycosylation. Genes to Cells, 2(2), 129-141.