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


CONCLUSIONS
tsBN7 cells have a defect in N-linked glycosylation caused by the loss of DAD1.
Makishima
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka, Japan.
Nakashima
T
T
Nagata-Kuno
K
K
Fukushima
K
K
Iida
H
H
Sakaguchi
M
M
Ikehara
Y
Y
Komiyama
S
S
Nishimoto
T
T
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005816:Genetic Complementation Test; D006031:Glycosylation / Q000187:drug effects; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012441:Saccharomyces cerevisiae; D014166:Transferases / Q000235:genetics / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D008565:Membrane Proteins; D014415:Tunicamycin; D003577:Cytochrome P-450 Enzyme System; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase